Pleiotrophin Receptor Research Articles

Pleiotrophin Enhances Clonal Growth and Long-Term Expansion of Human Embryonic Stem Cells

To identify additional growth factors for optimizing propagation of human embryonic stem cells (hESCs), we mined publicly available data sets for the transcriptomes of murine and human ESCs and feeder cells, thereby generating a list of growth factors and complementary receptors. We identified the major pathways previously reported to be important, as well as several new ones. One pathway is the Pleiotrophin (PTN)-Pleiotrophin receptor (PTPRZ1) axis. Murine fibroblasts secrete Ptn, whereas hESCs expressed PTPRZ1, which is downregulated upon differentiation. Depletion of PTPRZ1 resulted in decreased colony formation and lower recovery of hESCs. Supplementation of chemically defined medium for feeder-free propagation of hESCs with PTN allowed higher recovery of hESCs without loss of pluripotency. PTN-PTPRZ1 functions here predominantly via an antiapoptotic effect mediated in part by the activation of Akt. These findings reveal the underlying importance of PTN in hESC survival and its usefulness in the clonal manipulation and large-scale propagation of hESCs. Disclosure of potential conflicts of interest is found at the end of this article.

Pleiotrophin Promotes Myeloma Proliferation through the Activation of Known and Novel Receptors.

Myeloma survival and proliferation in the bone marrow (BM) depends on the expression of a variety of autocrine and paracrine growth factors, including IL-6, insulin-like growth factor I (IGF-I), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). We recently discovered an additional new autocrine myeloma growth factor, pleiotrophin (PTN). PTN is an 18kD heparin-binding protein normally expressed during early development and downregulated in adults, but aberrant PTN re-expression has been associated with a variety of aggressive solid tumors including neuroblastoma, glioma, melanoma and lung, breast and prostate cancers. We found that interference with PTN using a polyclonal anti-PTN antibody inhibited the proliferation of myeloma cells in vitro and in vivo by inducing cell cycle arrest but not apoptosis. To determine the mechanism by which PTN stimulates myeloma proliferation we analyzed the expression of the known PTN receptors, syndecans 1 (CD138) and 3, the anaplastic lymphoma kinase (ALK) and the receptor tyrosine phosphatase beta/zeta (RPTPβ/ζ) on myeloma cells. The expression of ALK and RPTPβ/ζ has not previously been investigated in any hematologic malignancy. In addition to syndecan 1 we found that a subset of myeloma cell lines and BM mononuclear cells (BMMCs) from myeloma patients are RPTPβ/ζ+ by RT-PCR, Western blot and flow cytometry. Myeloma cells, however, do not express ALK. RPTPβ/ζ inhibition by PTN binding leads to the accumulation of β-catenin, and downstream activation of Wnt, NF-κB, MAPK and Akt-mediated cell signals in cells from solid tumors, signaling pathways known to contribute to myeloma cell survival and proliferation. The myeloma cell line MM-1S and the SCID-hu myeloma model LAGλ-1 are both CD138+ but RPTPβ/ζ−. Nonetheless, the growth of these cells is inhibited by anti-PTN antibody. CD138 lacks cytoplasmic signaling motifs suggesting that additional novel receptors are required for PTN-stimulated cell growth in these cells. To continue to define PTN-mediated signaling in myeloma cells we compared the induction of PTN-regulated signaling pathways in RPTPβ/ζ+ RPMI 8226 cells compared to RPTPβ/ζ − MM-1s cells by phospho-protein Western blot. We found differences in PTN-stimulated tyrosine phosphorylation between RPTPβ/ζ+ and RPTPβ/ζ −cells. Specifically, we also discovered that RPTPβ/ζ+ RPMI 8226 cells activate the MAPK Erk1/2. In contrast, we found Akt, but not Erk1/2, to be activated by PTN in RPTPβ/ζ − MM-1S cells. We are now continuing our analysis of PTN signaling in these cells. We are using also identifying new PTN receptors on the surface of myeloma cells by affinity chromatography using biotinylated-PTN. These studies will provide potential new targets that should lead to the development of novel targeted anti-myeloma therapies.

Effects of pleiotrophin, a heparin-binding growth factor, on human primary and immortalized chondrocytes

Pleiotrophin (PTN) is a secreted heparin-binding peptide expressed in mesodermal and neuroectodermal cells during development, but rarely in adult tissues. In fetal and juvenile, but not in mature cartilage, PTN is abundant. Furthermore, PTN is re-expressed in chondrocytes in early stages of osteoarthritis (OA). Since little is known about the functions of PTN in cartilage, we investigated the occurrence of PTN receptors in human articular cartilage in situ and PTN effects on human primary and immortalized chondrocytes in vitro. Receptor expression and regulation was monitored by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. PTN effects and signal transduction were studied by electrophoretic mobility shift, Boyden chamber cell migration and proliferation assays, effects on gene expression by real time RT-PCR and that on nitric oxide (NO) by the Griess reaction. Of the putative PTN signaling receptors, immortalized and primary chondrocytes (pc) expressed the anaplastic lymphoma kinase (ALK), less the receptor-type protein tyrosine phosphatase zeta/beta (PTPzeta). ALK expression was upregulated upon ligand exposure. PTN stimulation activated the AP-1 (activator protein-1) transcription factor and altered gene expression. Prolonged stimulation induced PTN mRNA expression slightly, reduced vascular endothelial growth factor (VEGF) mRNA as well as NO production. Whereas mRNA expression of matrix metalloproteinases (MMPs) MMP-1 and MMP-13 was reduced, their inhibitors TIMP-1 and TIMP-2 were induced. Furthermore, PTN stimulated chondrocyte migration and proliferation. These results show that PTN is an autocrine growth factor in cartilage. We suggest that PTN may be involved in the clustering and proliferation of chondrocytes observed in the early stages of OA.

Expression of pleiotrophin and its receptors in hepatic cells and in activated hepatic stellate cells (HSCs)– Pleiotrophin in hepatic stellate cells

Pleiotrophin (PTN) is a heparin-binding protein and a growth factor with a broad spectrum of biological activities very similar to the Fibroblast Growth Factors (FGFs). FGFs and PTN promote the growth, survival, and migration of various cells, and play many roles in epithelial mesenchymal interactions during organogenesis. Particulary, expression of PTN has been implicated in the development and tissue repair of the liver. PTN interacts with several receptor proteins which may transduce PTN signaling in a cell type specific way. As PTN receptor has been proposed syndecan-3, receptor-tyrosine phosphatase (RPTP)zeta, anaplastic lymphoma kinase (ALK) and the Wnt co-receptors LRP5/6. Syndecan-3 and RPTPzeta have been proposed to be the receptor of PTN-induced neurite outgrowth, migration and survival of embryonic neurons. In this study we analysed the expression of PTN, the expression of the receptors ALK and LRP5/6 in rat liver cells, in activated rat hepatic stellate cells and in human and rat myofibroblasts. Ligand and receptor expression was investigated at the mRNA level using reverse transcription polymerase chain reaction. All analysed hepatic nonparenchymal cells including stellate cells, sinusoidal endothelial cells and Kupffer cells demonstrate PTN-, ALK- and LRP5/6- mRNA expression. Hepatocytes did not express the anaplastic lymphoma kinase. Interestingly, since ALK and PTN are implicated in tumor formation, ALK expression was also not detected in the hepatoma cell line HepG2. Since PTN prevents apoptosis in serum-starved NIH3T3 fibroblasts via activation of ALK at concentrations much lower than those needed to induce cell growth, the function of PTN in the diseased liver may mainly be its anti-apoptotic signaling in HSCs.

Midkine and pleiotrophin in neural development and cancer

The midkine (MK) family consists of only two members, namely heparin-binding growth factors MK and pleiotrophin (PTN). During embryogenesis, MK is highly expressed in the mid-gestational period, whereas PTN expression reaches the maximum level around birth. Both proteins are localized in the radial glial processes of the embryonic brain, along which neural stem cells migrate and differentiate. Zebrafish and Xenopus MK can induce neural tissues. In addition, deposits of MK and/or PTN are found in neurodegenerative diseases, such as Alzheimer's disease and multiple system atrophy. Both molecules are induced in reactive astrocytes by ischemic insults. In this context, it is interesting that LDL receptor-related protein is a receptor for MK and PTN, and this receptor has been implicated in the pathogenesis of Alzheimer's disease. MK and PTN share receptors, and show similar biological activities that include fibrinolytic, anti-apoptotic, mitogenic, transforming, angiogenic, and chemotactic ones. These activities explain how these molecules are involved in carcinogenesis. MK is detected in human carcinoma specimens from pre-cancerous stages to advanced stages. Strong expression of PTN is also detected in several carcinomas, although, in general, MK is expressed more intensely and in a wide range of carcinomas than PTN. The blood MK level is frequently elevated in advanced human carcinomas, decreases after surgical removal of the tumors, and is correlated with prognostic factors. Thus, it is a good market for evaluating the progress of carcinomas. Furthermore, antisense oligonucleotides for MK and ribozymes for PTN show anti-tumor activity. Therefore, MK and PTN are candidate molecular targets for therapy for human carcinomas.

Pleiotrophin exhibits a trophic effect on survival of dopaminergic neurons in vitro.

To understand what kind of trophic factors are up-regulated in dopamine (DA)-depleted striatum, we first analysed the up-regulation of mRNAs using a DNA microarray in DA-depleted striatum where DAergic inputs were denervated by 6-OHDA. We then investigated whether or not such trophic factors had an effect on cultured dopaminergic neurons. The microarray analysis revealed that pleiotrophin (PTN), glial-derived neurotopic factor (GDNF) and others were up-regulated in DA-depleted striatum. As PTN has been reported to have a wide range of trophic effects on neurons, we focused on the functional role of PTN in the present study. The increase in PTN mRNA was confirmed by Northern blotting at 1-3 weeks after the lesion, reaching a peak at 1 week. In embryonic day 15 mesencephalic neuron culture, PTN increased the number of tyrosine hydroxylase (TH) -positive neurons in a dose-dependent manner (125.2 +/- 2.0% of the control at 50 ng/mL), while a family protein, midkine (10 ng/mL) did not show any trophic effect (99.3 +/- 0.7%). In addition, the PTN effect on DAergic neurons was additive to the GDNF effect. As PTN did not increase the number of microtubule-associated protein-2 (MAP 2)-positive neurons or promote the proliferation of dopaminergic progenitors in a bromodeoxyuridine (BrdU) labelling study, the effect appeared to enhance the specific survival of dopaminergic neurons. Expression of PTN receptors (syndecan-3, PTP-zeta) was detected on the cultured mesencephalic neurons, and also up-regulated in DA-depleted striatum. The data indicate that PTN is up-regulated in DA-depleted striatum and exhibits a trophic effect specifically on the survival of cultured dopaminergic neurons.

Midkine Binds to Anaplastic Lymphoma Kinase (ALK) and Acts as a Growth Factor for Different Cell Types

Midkine (MK) is a developmentally regulated, secreted growth factor homologous to pleiotrophin (PTN). To investigate the potential role of MK in tumor growth, we expressed MK in human SW-13 cells and studied receptor binding, signal transduction, and activity of MK. The MK protein stimulates soft agar colony formation in vitro and tumor growth of SW-13 cells in athymic nude mice, as well as proliferation of human endothelial cells from brain microvasculature and umbilical vein (HUVEC) in the low ng/ml range. MK binds to anaplastic lymphoma kinase (ALK), the receptor for PTN, with an apparent K(d) of 170 pm in intact cells, and this receptor binding of MK is competed by PTN with an apparent K(d) of approximately 20 pm. Monoclonal antibodies raised against the extracellular ligand-binding domain of ALK inhibit ALK receptor binding of MK as well as MK-stimulated colony formation of SW-13 cells. Furthermore, MK stimulates ALK phosphorylation in WI-38 human fibroblasts and activates PI3-kinase and MAP kinase signal transduction in WI-38, HUVEC, neuroblastoma (SH SY-5Y) and glioblastoma (U87MG) cells that express the ALK protein. We conclude that MK can act as a growth, survival, and angiogenic factor during tumorigenesis and signals through the ALK receptor.

Midkine and pleiotrophin: two related proteins involved in development, survival, inflammation and tumorigenesis.

Midkine (MK) and pleiotrophin (PTN) are low molecular weight proteins with closely related structures. They are mainly composed of two domains held by disulfide bridges, and there are three antiparallel beta-sheets in each domain. MK and PTN promote the growth, survival, and migration of various cells, and play roles in neurogenesis and epithelial mesenchymal interactions during organogenesis. A chondroitin sulfate proteoglycan, protein-tyrosine phosphatase zeta (PTPzeta), is a receptor for MK and PTN. The downstream signaling system includes ERK and PI3 kinase. MK binds to the chondroitin sulfate portion of PTPzeta with high affinity. Among the various chondroitin sulfate structures, the E unit, which has 4,6-disulfated N-acetylgalactosamine, provides the strongest binding site. The expression of MK and PTN is increased in various human tumors, making them promising as tumor markers and as targets for tumor therapy. MK and PTN expression also increases upon ischemic injury. MK enhances the migration of inflammatory cells, and is involved in neointima formation and renal injury following ischemia. MK is also interesting from the viewpoints of the treatment of neurodegenerative diseases, increasing the efficiency of in vitro development, and the prevention of HIV infection.

Pleiotrophin Signaling through Anaplastic Lymphoma Kinase Is Rate-limiting for Glioblastoma Growth

Glioblastoma multiforme is the most common highly aggressive human brain cancer, and receptor tyrosine kinases have been implicated in the progression of this malignancy. We have recently identified anaplastic lymphoma kinase (ALK) as a tyrosine kinase receptor for pleiotrophin, a secreted growth factor that is highly expressed during embryonic brain development and in tumors of the central nervous system. Here we report on the contribution of pleiotrophin-ALK signaling to glioblastoma growth. We found ALK overexpressed in human glioblastoma relative to normal brain and detected ALK mRNA in glioblastoma cell lines. We reduced the endogenous ALK in glioblastoma cells by ribozyme targeting and demonstrated that this prevents pleiotrophin-stimulated phosphorylation of the anti-apoptotic protein Akt. Furthermore, this depletion of ALK reduced tumor growth of xenografts in athymic nude mice and prolonged survival of the animals because of increased apoptosis in the tumors. These findings directly implicate ALK signaling as a rate-limiting factor in the growth of glioblastoma multiforme and suggest potential utility of therapeutic targeting of ALK.

Identification of Anaplastic Lymphoma Kinase as a Receptor for the Growth Factor Pleiotrophin

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. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.

Pleiotrophin signals increased tyrosine phosphorylation of beta beta-catenin through inactivation of the intrinsic catalytic activity of the receptor-type protein tyrosine phosphatase beta/zeta.

Pleiotrophin (PTN) is a platelet-derived growth factor-inducible, 18-kDa heparin-binding cytokine that signals diverse phenotypes in normal and deregulated cellular growth and differentiation. To seek the mechanisms of PTN signaling, we studied the interactions of PTN with the receptor protein tyrosine phosphatase (RPTP) beta/zeta in U373-MG cells. Our results suggest that PTN is a natural ligand for RPTP beta/zeta. PTN signals through "ligand-dependent receptor inactivation" of RPTP beta/zeta and disrupts its normal roles in the regulation of steady-state tyrosine phosphorylation of downstream signaling molecules. We have found that PTN binds to and functionally inactivates the catalytic activity of RPTP beta/zeta. We also have found that an active site-containing domain of RPTP beta/zeta both binds beta-catenin and functionally reduces its levels of tyrosine phosphorylation when added to lysates of pervanidate-treated cells. In contrast, an (inactivating) active-site mutant of RPTP beta/zeta also binds beta-catenin but fails to reduce tyrosine phosphorylation of beta-catenin. Finally, in parallel to its ability to inactivate endogenous RPTP beta/zeta, PTN sharply increases tyrosine phosphorylation of beta-catenin in PTN-treated cells. The results suggest that in unstimulated cells, RPTP beta/zeta is intrinsically active and functions as an important regulator in the reciprocal control of the steady-state tyrosine phosphorylation levels of beta-catenin by tyrosine kinases and phosphatases. The results also suggest that RPTP beta/zeta is a functional receptor for PTN; PTN signals through ligand-dependent receptor inactivation of RPTP beta/zeta to increase levels of tyrosine phosphorylation of beta-catenin to initiate downstream signaling. PTN is the first natural ligand identified for any of the RPTP family; its identification provides a unique tool to pursue the novel signaling pathway activated by PTN and the relationship of PTN signaling with other pathways regulating beta-catenin.