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 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.

Pleiotrophin disrupts calcium-dependent homophilic cell-cell adhesion and initiates an epithelial-mesenchymal transition.

Regulation of the levels of tyrosine phosphorylation is essential to maintain the functions of proteins in different signaling pathways and other cellular systems, but how the steady-state levels of tyrosine phosphorylation are coordinated in different cellular systems to initiate complex cellular functions remains a formidable challenge. The receptor protein tyrosine phosphatase (RPTP)beta/zeta is a transmembrane tyrosine phosphatase whose substrates include proteins important in intracellular and transmembrane protein-signaling pathways, cytoskeletal structure, cell-cell adhesion, endocytosis, and chromatin remodeling. Pleiotrophin (PTN the protein and Ptn the gene) is a ligand for RPTPbeta/zeta; PTN inactivates RPTPbeta/zeta, leaving unchecked the continued endogenous activity of tyrosine kinases that increase phosphorylation of the substrates of RPTPbeta/zeta at sites dephosphorylated by RPTPbeta/zeta in cells not stimulated by PTN. Thus, through the regulation of the tyrosine phosphatase activity of RPTPbeta/zeta, the PTN/RPTPbeta/zeta signaling pathway coordinately regulates the levels of tyrosine phosphorylation of proteins in many cellular systems. We now demonstrate that PTN disrupts cytoskeletal protein complexes, ablates calcium-dependent homophilic cell-cell adhesion, stimulates ubiquitination and degradation of N-cadherin, reorganizes the actin cytoskeleton, and induces a morphological epithelial-mesenchymal transition (EMT) in PTN-stimulated U373 cells. The data suggest that increased tyrosine phosphorylation of the different substrates of RPTPbeta/zeta in PTN-stimulated cells alone is sufficient to coordinately stimulate the different functions needed for an EMT; it is possible that PTN initiates an EMT in cells at sites where PTN is expressed in development and in malignant cells that inappropriately express Ptn.

Platelet-derived growth factor (PDGF) receptor-alpha activates c-Jun NH2-terminal kinase-1 and antagonizes PDGF receptor-beta -induced phenotypic transformation.

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells. The PDGF B-chain (c-sis proto-oncogene) homodimer (PDGF BB) and v-sis, its viral counterpart, activate both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR) and mediate anchorage-independent growth in NIH3T3 cells. In contrast, the PDGF A chain homodimer (PDGF AA) activates alpha-PDGFR only and fails to induce phenotypic transformation. In the present study, we investigated alpha- and beta-PDGFR specific signaling pathways that are responsible for the differences between the transforming ability of PDGF AA and BB. To study PDGF BB activation of beta-PDGFR, we established NIH3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an antisense construct of alpha-PDGFR. Here, we demonstrate that beta-PDGFR activation alone is sufficient for PDGF BB-mediated anchorage-independent cell growth. More importantly, inhibition of alpha-PDGFR signaling enhanced PDGF BB-mediated phenotypic transformation, suggesting that alpha-PDGFR antagonizes beta-PDGFR-induced transformation. While both alpha- and beta-receptors effectively activate ERKs, alpha-PDGFR, but not beta-PDGFR, activates stress-activated protein kinase-1/c-Jun NH(2)-terminal kinase-1 (JNK-1). Inhibition of JNK-1 activity using a dominant-negative JNK-1 mutant markedly enhanced PDGF BB-mediated anchorage-independent cell growth, demonstrating an antagonistic role for JNK-1 in PDGF-induced transformation. Consistently, overexpression of wild-type JNK-1 reduced PDGF BB-mediated transformation. Taken together, the present study showed that alpha- and beta-PDGFRs differentially regulate Ras-mitogen-activated protein kinase pathways critical for regulation of cell transformation, and transformation suppressing activity of alpha-PDGFR involves JNK-1 activation.

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.

A dominant-negative pleiotrophin mutant introduced by homologous recombination leads to germ-cell apoptosis in male mice.

Pleiotrophin (PTN) is an 18-kDa heparin-binding secretory growth/differentiation factor for different cell types. Its gene is differentially expressed in both mesenchyme and central nervous system during development and highly expressed in a number of different human tumors. Recently, a PTN mutant was found to act as a dominant-negative effector of PTN signaling. We have now used homologous recombination to introduce the dominant-negative PTN mutant into embryonic stem cells to generate chimeric mice. All highly chimeric male mice with germinal epithelium exclusively derived from embryonic stem cells with the heterologous PTN mutation were sterile. Their testes were uniformly atrophic, and the spermatocytes were strikingly apoptotic at all stages of development. The results support a central role of PTN signaling in normal spermatogenesis and suggest that interruption of PTN signaling may lead to sterility in males.

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 and midkine, a family of mitogenic and angiogenic heparin-binding growth and differentiation factors.

The heparin-binding polypeptide homologs pleiotrophin and midkine are the only known members of a family of secreted growth/differentiation cytokines. Pleiotrophin and midkine are both developmentally regulated and highly conserved among species. They signal a number of physiological functions involved with angiogenesis, neuorogenesis, cell migration, and mesoderm-epithelial interactions. Constitutive expression of pleiotrophin and midkine in responsive cells support their role as "tumor growth factors" and positive regulators of tumor angiogenesis. Widespread deregulation of pleiotrophin and midkine is found in many known human cancers or their derived cell lines, and the molecular targeting of pleiotrophin to block its signaling in tumor cells has limited tumor growth and metastasis in animal models. Elucidating the molecular mechanisms of pleiotrophin and midkine action in tumorgenesis and tumor angiogenesis may lead to the identification of novel targets for tumor therapy.

Imbalanced expression of functionally different WT1 isoforms may contribute to sporadic unilateral Wilms' tumor.

Functional loss of the product of the Wilm's tumor suppressor gene (wt1) has been identified in subsets of familial Wilms' tumors. Previously, four alternative splice products of WT1 were recognized and each was found to regulate transcription of effector genes differently, suggesting that disruption of the normal ratio of these spliced products will disrupt the normal expression patterns of WT1 effector genes and perhaps lead to Wilms' tumor. In support of these suggestions, we found that four of seven cases of sporadic unilateral Wilms' tumor had striking differences in the ratios of the spliced products of WT1 compared with each other and normal kidney. These data indicate that in addition to structural mutations, alterations in the relative amounts of the mature WT1 isoforms may also be important in the etiology of sporadic Wilms' tumor.

Wilms' tumor gene product WT1 arrests macrophage differentiation of HL-60 cells through its zinc-finger domain.

Wilms' tumor is associated with mutations of WT1, a zinc-finger transcription factor that is essential for the development of the metanephric kidney and the urogenital system. High levels of WT1 expression also have been detected in myeloid leukemia cells, suggesting that WT1 may be important in other neoplasms as well. To seek a role of high level expression of WT1 in the differential arrest characteristic of myeloid leukemia, WT1 or its zinc-finger domain alone was stably expressed in human promyeloid leukemia (HL-60) cells and the ability of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to induce macrophage differentiation was examined. HL-60 cell differentiation was completely arrested in TPA treated cells that expressed WT1 or its zinc-finger domain alone whereas TPA fully induced macrophage differentiation in control HL-60 cells, indicating that high level expression of WT1 is capable of differentiation arrest of myeloid cells and that its effect may be mediated through its zinc-finger domain. To determine if the zinc-finger domain of WT1 directly influences transcription, it was brought to promoter DNA as a fusion protein with the Gal4 DNA binding domain. The fusion protein failed to regulate transcription of a reporter gene but when the zinc-finger domain of WT1 was brought to DNA with a promoter containing two upstream WT1-binding sites, reporter gene expression was activated approximately threefold, suggesting that WT1 interferes with myeloid differentiation through the ability of its zinc-finger domain to compete with other transcription factors for common promoter elements.

Upregulation of pleiotrophin gene expression in developing microvasculature, macrophages, and astrocytes after acute ischemic brain injury.

Pleiotrophin (PTN) is a heparin-binding, 18 kDa secretory protein that functions to induce mitogenesis, angiogenesis, differentiation, and transformation in vitro. PTN gene (Ptn) expression is highly regulated during development and is highest at sites in which mitogenesis, angiogenesis, and differentiation are active. In striking contrast, with the exception of the neuron, the Ptn gene is only minimally expressed in adults. We now demonstrate that Ptn gene expression is strikingly upregulated within 3 d in OX42-positive macrophages, astrocytes, and endothelial cells in areas of developing neovasculature after focal cerebral ischemia in adult rat. Ptn gene expression remains upregulated in these same cells and sites 7 and 14 d after ischemic injury. However, expression of the Ptn gene is significantly decreased in cortical neurons 6 and 24 hr after injury and is undetectable in degenerating neurons at day 3. Neurons in contralateral cortex continue to express Ptn in levels equal to control, uninjured brain. It is suggested that PTN may have a vital role in neovascular formation in postischemic brain and that postischemic brain is an important model in which to analyze sequential gene expression in developing neovasculature. In contrast, Ptn gene expression in injured neurons destined not to recover is strikingly reduced, and potentially its absence may contribute to the failure of the neuron to survive.

Human breast cancer growth inhibited in vivo by a dominant negative pleiotrophin mutant.

Pleiotrophin (PTN) is a recently described 18- kDa heparin binding growth/differentiation factor. It also is a proto-oncogene; cells transformed by the Ptn gene form highly angiogenic tumors when implanted into the nude mouse. PTN may be an important regulator of transformation in other tumors, because constitutively high levels of expression of the pleiotrophin (Ptn) gene are found in human breast cancer and other malignant cell lines, and its levels of expression are high in many human tumor specimens. To determine whether PTN is an important regulator of the malignant phenotype of human breast cancer cells, we constructed a mutant cDNA to encode a truncated PTN designed to heterodimerize with the product of the endogenous Ptn gene during processing. The mutant gene product blocked transformation of NIH 3T3 cells by the wild type (wt) Ptn gene product. The mutant Ptn cDNA was then introduced into human breast cancer MDA-MB-231 cells, and clonal lines that stably express the mutant Ptn cDNA were selected. The truncated PTN was shown to form heterodimers with the endogenous Ptn gene product in these cells. Furthermore, the MDA-MB-231 cells that express the mutant Ptn gene were no longer transformed; they failed to form plaques or colonies in soft agar and were unable to form tumors in the athymic nude mouse. The results establish an important role of PTN in the dysregulated growth of human breast cancer cells and suggest that constitutive expression of PTN may be essential to the malignant phenotype of human breast cancers in vivo.

Transcription regulation of the PDGF A-chain gene by first intron elements.

A cis-acting regulatory region within the first intron of the human platelet-derived growth factor (PDGF) A-chain gene has been identified that functions to negatively regulate transcription of PDGF A-chain promoter/CAT reporter constructs in both A172 and HeLa cells and that functions independent of position, orientation, and promoter context. Further dissection of this region revealed several independently acting negative regulatory elements that exhibited cell-type specificity. These results suggest that the first intron of the PDGF A-chain gene contains negative regulatory elements that may cooperate to regulate the cell-type specific expression of the PDGF A-chain gene.

Localization of pleiotrophin and its mRNA in subpopulations of neurons and their corresponding axonal tracts suggests important roles in neural-glial interactions during development and in maturity.

Trophic factors are being increasingly recognized as important contributors to growth, differentiation, and maintenance of viability within the mammalian nervous system during development. Pleiotrophin (PTN) is a secreted 18-kDa heparin binding protein that stimulates mitogenesis and angiogenesis and neurite and glial process outgrowth guidance activities in vitro. We localized the sites and time course of expression of the Ptn gene and its protein product in developing and adult mouse nervous system. Expression of the Ptn gene was first observed at embryo day 8.5 (E8.5). At E12.5, transcripts of the Ptn gene were localized in developing neuroepithelium at sites of active cell division in the spinal cord and brain. At E15.5, transcripts were found in the somata of some but not all neurons and glia whereas in the adult its pattern of expression was nearly exclusively restricted to the brain. The PTN protein was found almost entirely in association with the axonal tracts during development and in adults. Furthermore, as opposed to the finding of PTN in both central and peripheral nervous systems during development, PTN was not expressed beyond the exit where axonal tracts become the peripheral nervous system in adults. At all sites and times examined, the somata that contained Ptn transcripts corresponded with the axonal tracts that contained the PTN protein. The results establish that Ptn is expressed in early development at sites of active mitogenesis in developing neuroepithelium and later in both glial cells and neurons at sites of neuronal and glial process formation in developing axonal tracts. The findings establish a correspondence in the localization of PTN within the nervous system at sites of normal developmental processes that correlate with the functional activities of PTN previously described in vitro.

Molecular cloning of the cDNA and chromosome localization of the gene for human ubiquitin-conjugating enzyme 9.

We report a novel human gene whose product specifically associates with the negative regulatory domain of the Wilms' tumor gene product (WT1) in a yeast two-hybrid screen and with WT1 in immunoprecipitation and glutathione S-transferase (GST) capture assays. The gene encodes a 17-kDa protein that has 56% amino acid sequence identity with yeast ubiquitin-conjugating enzyme (yUBC) 9, a protein required for cell cycle progression in yeast, and significant identity with other subfamilies of ubiquitin-conjugating enzymes. The human gene fully complements yeast that have a temperature-sensitive yUBC9 gene mutation to fully restore normal growth, indicating that we have cloned a functionally conserved human (h) homolog of yUBC9. Transcripts of hUBC9 of 4.4 kilobases (kb), 2.8 kb, and 1.3 kb were found in all human tissues tested. A single copy of the hUBC9 gene was found and localized to human chromosome 16p13.3. We conclude that hUBC9 retains striking structural and functional conservation with yUBC9 and suggest a possible link of the ubiquitin/proteosome proteolytic pathway and the WT1 transcriptional repressor system.

Midkine gene and the m4 muscarinic acid receptor gene are linked on mouse chromosome 2.

The retinoid acid inducible midkine gene encodes a highly basic, heparin-binding protein that possesses potential functions of growth or differentiation during the early stages of mouse embryonic development. Midkine gene has been mapped to mouse Chromosome 2. Here we report that the midkine gene is located in close proximity to the m4 muscarinic acid receptor gene. M4 codes for a transmembrane G protein that functions to transduce the action of acetylcholine neural transmitters in the nervous system. The two genes have the directions of their transcriptions running toward one another, and the distance between their respective 3' ends is approximately 1.3 kb.

Platelet-derived growth factor induces apoptosis in growth-arrested murine fibroblasts.

The platelet-derived growth factor (PDGF) is a potent mitogen for murine fibroblasts. PDGF-stimulated cells express a set of immediate-early-response genes but require additional (progression) factors in serum to progress through the cell cycle. Serum-deprived cells are reversibly arrested in G0 phase and fail to fully traverse the G1 phase of the cell cycle when stimulated by PDGF alone. We now report that serum-deprived normal rat kidney fibroblast (NRK) cells stimulated by either PDGF AA or PDGF BB homodimers undergo apoptotic cell death. Furthermore, we show that epidermal growth factor also induces apoptotic cell death in serum-deprived NRK cells, epidermal growth factor enhances the rate of apoptosis in PDGF-treated cells, and a progression factor (insulin) but not endogenously expressed Bc1-2 fully protects NRK cells from PDGF-stimulated apoptosis. The results indicate that PDGF induces apoptosis in growth-arrested NRK cells and that the inability of NRK cells to transit the G1/S checkpoint is the critical determinant in establishing the genetic program(s) to direct the PDGF signal to apoptosis. The results suggest that polypeptide growth factors in vivo may signal cell fate positively or negatively in settings that limit the potential of cells to completely transit the cell cycle.

Differential expression of pleiotrophin and midkine in advanced neuroblastomas.

Pleiotrophin (PTN) and midkine (MK) are members of a new family of neurotrophic factors whose expression is developmentally regulated. PTN also transforms NIH 3T3 cells, and MK is mitogenic to certain cell lines. Neuroblastomas are tumors derived from neural crest cells, and recent studies have revealed that the biology of these tumors is at least partly regulated by neurotrophic factors and their receptors. To examine the expression of PTN and MK in neuroblastoma, we analyzed their mRNA expression in 72 primary neuroblastomas and 11 neuroblastoma cell lines as well as other tissues and cell lines. PTN is highly expressed in favorable neuroblastomas (stages I, II, and IV-S, n = 44), whereas it is expressed at a significantly lower level in advanced tumors (stages III and IV, n = 28, P = 0.003). PTN is not expressed in either aggressive neuroblastomas with N-myc amplification or in neuroblastoma cell lines. Moreover, the expression pattern of PTN was similar to that of TRK-A, the high affinity receptor for nerve growth factor, in that it is correlated with a favorable prognosis (P < 0.004). In contrast, MK is highly expressed in almost all primary neuroblastomas and cell lines and showed no correlation with disease stage or N-myc amplification. These results suggest that differential expression of PTN and MK may have an important role in regulating growth and differentiation of neuroblastomas.

WT1, the Wilms' tumor suppressor gene product, represses transcription through an interactive nuclear protein.

The Wilms' tumor suppressor gene, wt1, encodes a transcription factor of the zinc finger family. Mutations in WT1 have been detected in subsets of Wilms' tumor and in patients with the Denys-Drash Syndrome. In order to determine how WT1 regulates transcription and perhaps the consequences that mutations in WT1 may have, we established that residues 85-124 and 181-250 of WT1 constitute domains that function independently with a DNA binding domain to repress or activate transcription, respectively, and function equally effectively with heterologous promoters, suggesting the activator and repressor domains interact with nuclear components of general importance. To seek evidence for such components, increasing concentrations of WT1 repressor domain without a zinc finger DNA binding domain were co-transfected with fixed concentrations of wild-type (wt) WT1 and PDGF A-chain promoter/reporter gene constructs. As levels of the repressor domain were increased, a progressive loss of wt WT1 repressor activity and a progressive increase in its activation were observed, suggesting that the repressor domain of WT1 competes with wt WT1 for an interactive protein that is an essential component of the repressor activity of wt WT1. Because the most common mutation associated with Denys-Drash Syndrome disrupts the zinc finger domains of WT1, the results also suggest that the mutant WT1 may have aberrant DNA binding activity and perhaps function as a dominant negative effector of wt WT1.