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.     

Anti-apoptotic signaling of pleiotrophin through its receptor,anaplastic lymphoma kinase

The secreted growth factor pleiotrophin (PTN) can induce mitogenesis in cells that express the receptor for this growth factor, anaplastic lymphoma kinase (ALK). Here we examine the ability of PTN to produce anti-apoptotic signals. We demonstrate that PTN is a survival factor for SW-13 epithelial cells and show that ribozyme-mediated depletion of ALK from SW-13 cells abolishes this effect of PTN. Furthermore, in serum-starved NIH3T3 fibroblasts PTN prevents apoptosis (measured by annexin V staining) with an EC(50) of 0.2 ng/ml and induces cell growth at higher concentrations of PTN. A polyclonal antibody against the PTN ligand-binding domain of the ALK receptor (alpha-LBD) was a partial agonist for ALK in NIH3T3 cells. This alpha-LBD antibody showed high agonist activity for anti-apoptosis (56 +/- 9% relative to PTN), low agonist activity for cell growth (21 +/- 1% relative to PTN), and was an antagonist of PTN-induced cell growth (61 +/- 2% inhibition). Both MAP kinase and phosphatidylinositol (PI) 3-kinase cascades in NIH3T3 cells were activated by PTN, and this effect persisted for up to 3 h. Surprisingly, the anti-apoptotic effect of PTN was completely blocked by the MAP kinase inhibitor UO126, but was not affected by the PI 3-kinase inhibitor LY294002. In contrast, PTN-dependent cell growth required both MAPK and PI 3-kinase activity. We conclude that anti-apoptotic signaling of PTN through ALK in NIH3T3 fibroblasts is via the MAP kinase pathway.     

Pleiotrophin stimulates fibroblasts and endothelial and epithelial cells and is expressed in human cancer.

Previously we reported the purification of the heparin-binding growth factor pleiotrophin (PTN) from supernatants of the human breast cancer cell line MDA-MB-231. To investigate further the biological activities of PTN and its potential role in cancer, we cloned a PTN cDNA and expressed the gene in a human kidney and in a human adrenal carcinoma cell line (SW-13). The supernatants harvested from cells transfected with PTN contained a heparin-binding specific protein of an apparent molecular mass of 18 kDa. These supernatants stimulated the proliferation of endothelial cells as well as the anchorage-independent growth of SW-13 cells and of normal rat kidney fibroblasts. Furthermore, SW-13 cells transfected with PTN acquired autonomous growth in soft agar and were tumorigenic in athymic nude mice. In contrast to these results with PTN from human cells, PTN obtained from insect cells (Sf9) using recombinant baculovirus as a vector was biologically inactive. We detected high levels of PTN mRNA in 16 of 27 primary human breast cancer samples (62%) as well as in 8 of 8 carcinogen-induced rat mammary tumors. Furthermore, 9 of 34 human tumor cell lines of different origin showed detectable PTN mRNA. We conclude that PTN may function as a tumor growth and angiogenesis factor in addition to its role during embryonic development.     

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.     

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.     

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.     

Anaplastic lymphoma kinase is activated through the pleiotrophin/receptor protein-tyrosine phosphatase beta/zeta signaling pathway: an alternative mechanism of receptor tyrosine kinase activation

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) first discovered as the constitutively active nucleophosmin-ALK oncoprotein in anaplastic large cell lymphomas (ALCL). Full-length ALK has a critical role in normal development and differentiation. Activated full-length ALK also is found in different malignant cancers. Nevertheless, the ligand to activate ALK remained unknown until recently, when ALK was proposed to be the physiological receptor of the cytokine pleiotrophin (PTN, Ptn). However, earlier studies had demonstrated that receptor protein tyrosine phosphatase (RPTP) beta/zeta is a physiological PTN receptor. We now demonstrate that phosphorylation of ALK in PTN-stimulated cells is mediated through the PTN/RPTPbeta/zeta signaling pathway. ALK is phosphorylated independently of a direct interaction of PTN with ALK. The data thus support a unique model of ALK activation. In cells not stimulated by PTN, RPTPbeta/zeta dephosphorylates ALK at the site(s) in ALK that is undergoing autophosphorylation through autoactivation. In contrast, when RPTPbeta/zeta is inactivated in PTN-stimulated cells, the sites that are autophosphorylated in ALK no longer can be dephosphorylated by RPTPbeta/zeta; thus, autoactivation and tyrosine phosphorylation of ALK rapidly increase. The data indicate that the PTN/RPTPbeta/zeta signaling pathway is a critical regulator of the steady state levels of tyrosine phosphorylation and activation of ALK; the data support the conclusion that ALK phosphorylation and activation in PTN-stimulated cells are increased through a unique "alternative mechanism of RTK activation."     

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.     

Anaplastic lymphoma kinase proteins in growth control and cancer

The normal functions of full-length anaplastic lymphoma kinase (ALK) remain to be completely elucidated. Although considered to be important in neural development, recent studies in Drosophila also highlight a role for ALK in gut muscle differentiation. Indeed, the Drosophila model offers a future arena for the study of ALK, its ligands and signalling cascades. The discovery of activated fusion forms of the ALK tyrosine kinase in anaplastic large cell lymphoma (ALCL) has dramatically improved our understanding of the pathogenesis of these lymphomas and enhanced the pathological diagnosis of this subtype of non-Hodgkin's lymphoma (NHL). Likewise, the realisation that a high percentage of inflammatory myofibroblastic tumours express activated-ALK fusion proteins has clarified the causation of these mesenchymal neoplasms and provided for their easier discrimination from other mesenchymal-derived inflammatory myofibroblastic tumour (IMT) mimics. Recent reports of ALK expression in a range of carcinoma-derived cell lines together with its apparent role as a receptor for PTN and MK, both of which have been implicated in tumourigenesis, raise the possibility that ALK-mediated signalling could play a role in the development and/or progression of a number of common solid tumours. The therapeutic targeting of ALK may prove to have efficacy in the treatment of many of these neoplasms.     

Autocrine growth factors for human tumor clonogenic cells

A human epithelial-derived cell line, SW-13, releases a soluble substance that functions as an autocrine growth factor. SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, form a few small colonies when suspended in soft agar at low densities. The number of colonies increased significantly when either viable SW-13 cells or serum-free medium conditioned by SW-13 cells (CM) was added to agar underlayers. CM increased colony formation in a dose-dependent fashion. Clonal growth at low cell densities was dependent on the presence of both horse serum and SW-13 CM. Neither activity alone was capable of sustaining growth. Even when cells were plated at high densities CM could not substitute for serum, but could reduce the threshold serum concentration. The results suggest that autocrine and serum-derived factors act in concert to maintain clonal growth of epithelial tumor cells in soft agar.     

Differential induction of glioblastoma migration and growth by two forms of pleiotrophin

Glioblastoma is the most common malignant brain tumor of adults and one of the most lethal cancers. The secreted growth factor pleiotrophin (PTN) promotes glioblastoma migration and proliferation, initiating its oncogenic activities through two cell surface receptors, the protein tyrosine phosphatase receptor zeta (PTPRZ1) and the anaplastic lymphoma kinase (ALK), respectively. Here, we report on the presence and purification of two naturally occurring forms of PTN (18 and 15 kDa) that differentially promote glioblastoma migration and proliferation. Using a panel of glioblastoma cell lines, including low passage patient-derived cultures, we demonstrate that PTN15 promotes glioblastoma proliferation in an ALK-dependent fashion, whereas immobilized PTN18 promotes haptotactic migration of glioblastoma cells in a PTPRZ1-dependent fashion. Mass spectrometric analysis indicated that PTN15 differs from PTN18 by processing of 12 C-terminal amino acids. To demonstrate clinical relevance, we show that PTN15, PTN18, and PTPRZ1 are significantly overexpressed in glioblastoma relative to normal brain at both mRNA and protein levels using microarray, Western blot, and tissue microarray analyses on human tumors. These results indicate that the PTN18-PTPRZ1 and the PTN15-ALK signaling pathways represent potentially important therapeutic targets for glioblastoma invasion and growth.     

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.     

Restoration of transforming growth factor-beta type II receptor reduces tumorigenicity in the human adrenocortical carcinoma SW-13 cell line.

Transforming growth factor-beta (TGF-beta) is a potent growth suppressor. Acquisition of TGF-beta resistance has been reported in many tumors, and has been associated with reduced TGF-beta receptor expression. In this study, we examined TGF-beta 1, TGF-beta type I receptor (TbetaRI) and TGF-beta type II receptor (TbetaRII) expression in SW-13 adrenocortical carcinoma cells by Northern and Western blot analysis. SW-13 cells did not express TbetaRII mRNA or protein. We have investigated the role of TbetaRII in modulating tumorigenic potential using stably transfected SW-13 cells with TbetaRII expression plasmid. TbetaRII-positive SW-13 cell growth was inhibited by exogenous human TGF-beta1 (hTGF-beta1) in a dose-dependent manner. In contrast, SW-13 cells and control clones transfected with empty vector remained hTGF-beta1-insensitive. Xenograft examination in athymic nude mice demonstrated that TbetaRII-positive SW-13 cells reduced tumor-forming activity. Reconstructing the TbetaRII can lead to reversion of the malignant phenotype of TbetaRII-negative human adrenocortical carcinoma, which contains SW-13 cells. Reduced TbetaRII expression may play a critical role in determining the malignant phenotype of human adrenocortical carcinoma.     

Anaplastic lymphoma kinase: “Ligand Independent Activation” mediated by the PTN/RPTPβ/ζ signaling pathway

Anaplastic lymphoma kinase is essential in early development, differentiation, and maintenance of cell survival; nevertheless, the mechanism to activate ALK has remained elusive. ALK has remained an “Orphan Receptor.” The studies cited below describe a unique mechanism termed “Ligand Independent Activation.” It is shown that activation of ALK results when the cytokine pleiotrophin (PTN) interacts with its receptor, the receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ). Pleiotrophin inactivates the catalytic activity of RPTPβ/ζ, which, when not inactivated, dephosphorylates phosphotyrosine sites in the activation domain of ALK; as a consequence of the inactivation of RPTPβ/ζ by PTN, autophosphorylation and autoactivation of ALK rapidly follow. The PTN/RPTPβ/ζ signaling pathway thus regulates the catalytic activity of ALK and tyrosine phosphorylation levels of ALK downstream target proteins. Furthermore, since ALK is only one of the key ALK phosphoproteins targeted by the PTN/RPTPβ/ζ signaling pathway, the PTN/RPTPβ/ζ signaling pathway has the potential to coordinately regulate tyrosine phosphorylation of other different key proteins in multiple cellular compartments. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Inhibition of GROalpha-induced human endothelial cell proliferation by the alpha-chemokine inhibitor antileukinate

GROalpha, an autocrine mitogenic factor for melanoma cell lines, belongs to the superfamily of alpha-chemokines. Here, we report that GROalpha stimulates the growth of human umbilical vein endothelial cells (HUVEC) in vitro, with proliferation being significantly stimulated by 100 nM recombinant human (rh) GROalpha. Proliferation was significantly inhibited by 100 microg/ml anti- human GROalpha monoclonal antibody (mAb), while excess GROalpha restored the growth. The addition of rhIL-8, rhIP-10, anti-human IL-8 or anti-human ENA-78 mAbs did not alter HUVEC proliferation. [125I]IL-8 binding to HUVEC was saturable and inhibited by non-radioactively iodinated IL-8, but not non-iodinated IL-8. [125I]GROalpha binding was also inhibited by iodinated IL-8. Since these data suggested specific binding sites for alpha-chemokines on HUVEC, we tested the effect of antileukinate, a potent alpha-chemokine receptor inhibitor, on [125I]GROalpha binding. Antileukinate inhibited GROalpha binding and suppressed HUVEC proliferation in a dose-dependent manner. Antileukinate was not cytotoxic, with no decrease in cell viability in the presence of 100 microM antileukinate. These findings suggest that GROalpha is essential for HUVEC growth factor and that antileukinate inhibits growth by preventing autocrine GROalpha receptor binding. This raises the interesting possibility of alpha-chemokine receptor inhibitors, such as antileukinate, in the treatment of cancer where angiogenesis is an important factor for tumour growth.     

In situ hybridization analysis of the temporospatial expression of the midkine/pleiotrophin family in rat embryonic pituitary gland

Pituitary gland development is controlled by numerous signaling molecules, which are produced in the oral ectoderm and diencephalon. A newly described family of heparin-binding growth factors, namely midkine (MK)/pleiotrophin (PTN), is involved in regulating the growth and differentiation of many tissues and organs. Using in situ hybridization with digoxigenin-labeled cRNA probes, we detected cells expressing MK and PTN in the developing rat pituitary gland. At embryonic day 12.5 (E12.5), MK expression was localized in Rathke’s pouch (derived from the oral ectoderm) and in the neurohypophyseal bud (derived from the diencephalon). From E12.5 to E19.5, MK mRNA was expressed in the developing neurohypophysis, and expression gradually decreased in the developing adenohypophysis. To characterize MK-expressing cells, we performed double-staining of MK mRNA and anterior pituitary hormones. At E19.5, no MK-expressing cells were stained with any hormone. In contrast, PTN was expressed only in the neurohypophysis primordium during all embryonic stages. In situ hybridization clearly showed that MK was expressed in primitive (immature/undifferentiated) adenohypophyseal cells and neurohypophyseal cells, whereas PTN was expressed only in neurohypophyseal cells. Thus, MK and PTN might play roles as signaling molecules during pituitary development.     

STAT1 is involved in signal transduction in the EPO induced HEL cells

Erythropoietin(EPO) is the major regulator of mamalian erythropoisis,which stimulates the growth and differentiation of hematopoietic cells through interaction with its receptor(EPO-R),Here we use HEL cells (a human erythro-leukemia cell line) as a model to elucidate the pathway of signal transduction in the EPO-induced HEL cells.Our data show that the EPOR (EPO receptor) on the surface of HEL cells interacts with the Janus tyrosine protein kinase(Jak2) to transduce intracellular signals through phosphorylation of cytoplasmic proteins in EPO-treated HEL cells.Both STAT1 and STAT5 in this cell line are tyrosine-phosphorylated and translocated to nucleus following the dinding of EPO to HEL cells.Furthermore,the dinding of both STAT1 and STAT5 proteins to specific DNA elements(SIE and PIE elements) is revealed in an EPO-dependent manner,Our data demonstrate that the pathway of signal transduction following the binding of EPO to HEL cells is similar to immature eryhroid cell from the spleen of mice infected with anemia strain of Friend virus.     

Specific binding of [3H]heparin to human carcinoma SW-13 and other mammalian cells

This study reports on the specific binding of [3H]heparin to human adrenocortical carcinoma cell line SW-13. Heparin binding to SW-13 cells is specific, saturable, and time- and temperature-dependent with maximum binding occurring between 90 and 120 min at 22 degrees C. Scatchard analysis revealed two classes of binding sites. The apparent Kd for high-affinity receptors is 2.14 x 10(-8) M with 1.48 x 10(6) sites per cells. Six other tested mammalian cell lines also have specific binding sites for heparin.     

Chondroitin sulfate chains on syndecan-1 and syndecan-4 from normal murine mammary gland epithelial cells are structurally and functionally distinct and cooperate with heparan sulfate chains to bind growth factors. A novel function to control binding of midkine, pleiotrophin, and basic fibroblast growth factor

A comparative analysis was carried out of heparan sulfate (HS) and chondroitin sulfate (CS) chains of the ectodomains of hybrid type transmembrane proteoglycans, syndecan-1 and -4, synthesized simultaneously by normal murine mammary gland epithelial cells. Although the HS chains were structurally indistinguishable, intriguingly the CS chains were structurally and functionally distinct, probably reflecting the differential regulation of sulfotransferases involved in the synthesis of HS and CS. The CS chains of the two syndecans comprised nonsulfated, 4-O-, 6-O-, and 4,6-O-disulfated N-acetylgalactosamine-containing disaccharide units and were significantly different, with a higher degree of sulfation for syndecan-4. Functional analysis using a BIAcore system showed that basic fibroblast growth factor (bFGF) specifically bound only to the HS chains of both syndecans, whereas midkine (MK) and pleiotrophin (PTN) bound not only to the HS but also to the CS chains. Stronger binding of MK and PTN to the CS chains of syndecan-4 than those of syndecan-1 was revealed, supporting the structural and functional differences. Intriguingly, removal of the CS chains decreased the association and dissociation rate constants of MK, PTN, and bFGF for both syndecans, suggesting the simultaneous binding of these growth factors to both types of chains, producing a ternary complex that transfers the growth factors to the corresponding cell surface receptors more efficiently compared with the HS chains alone. The involvement of the core protein was also shown in the binding of MK and PTN to syndecan-1, suggesting the possibility of cooperation with the HS and/or CS chains in the binding of these growth factors and their delivery to the cell surface receptors.