Receptor for advanced glycation end products (RAGE) mediates neurite outgrowth in vitro on amphoterin-coated substrates. Ligation of RAGE by two other ligands, advanced glycation end products or amyloid ␤-peptide, is suggested to play a role in cell injury mechanisms involving cellular oxidant stress and activation of the transcription factor NF-B. However, the RAGE signaling pathways in neurite outgrowth and cell injury are largely unknown. Here we show that transfection of RAGE to neuroblastoma cells induces extension of filopodia and neurites on amphoterin-coated substrates. Furthermore, ligation of RAGE in transfected cells enhances NF-B-dependent transcription. Both the RAGEmediated neurite outgrowth and activation of NF-B are blocked by deletion of the cytoplasmic domain of RAGE.Moreover, dominant negative Rac and Cdc42 but not dominant negative Ras inhibit the extension of neurites induced by RAGE-amphoterin interaction. In contrast, the activation of NF-B is inhibited by dominant negative Ras but not Rac or Cdc42. These data suggest that distinct signaling pathways are used by RAGE to induce neurite outgrowth and regulate gene expression through NF-B.

Bone has an enormous capacity for growth, regeneration, and remodeling. This capacity is largely due to induction of osteoblasts that are recruited to the site of bone formation. The recruitment of osteoblasts has not been fully elucidated, though the immediate environment of the cells is likely to play a role via cell– matrix interactions. We show here that heparin-binding growth-associated molecule (HB-GAM), an extracellular matrix–associated protein that enhances migratory responses in neurons, is prominently expressed in the cell matrices that act as target substrates for bone formation. Intriguingly, N-syndecan, which acts as a receptor for HB-GAM, is expressed by osteoblasts/osteoblast precursors, whose ultrastructural phenotypes suggest active cell motility. The hypothesis that HB-GAM/N-syndecan interaction mediates osteoblast recruitment, as inferred from developmental studies, was tested using osteoblast-type cells that express N-syndecan abundantly. These cells migrate rapidly to HB-GAM in a haptotactic transfilter assay and in a migration assay where HB-GAM patterns were created on culture wells. The mechanism of migration is similar to that previously described for the HB-GAM–induced migratory response of neurons. Our hypothesis that HB-GAM/N-syndecan interaction participates in regulation of osteoblast recruitment was tested using two different in vivo models: an adjuvant-induced arthritic model and a transgenic model. In the adjuvant-induced injury model, the expression of HB-GAM and of N-syndecan is strongly upregulated in the periosteum accompanying the regenerative response of bone. In the transgenic model, the HB-GAM expression is maintained in mesenchymal tissues with the highest expression in the periosteum. The HB-GAM transgenic mice develop a phenotype characterized by an increased bone thickness. HB-GAM may thus play an important role in bone formation, probably by mediating recruitment and attachment of osteoblasts/osteoblast precursors to the appropriate substrates for deposition of new bone.

No abstract

SUMMARYWe describe a nonradioactive preembedding in situ hybridization protocol using digoxigenin-labeled RNA probes and tyramide signal amplification to increase the sensitivity of detection. The protocol is sensitive enough for electron microscopic localization of endogenous messenger RNAs encoding ␤ -actin and amphoterin. Three visualization methods were compared: diaminobenzidine enhanced by nickel, Nanogold enhanced by silver and gold toning, and fluorescently labeled tyramides. Diaminobenzidine and Nanogold can be used in both light and electron microscopy. The nickel-enhanced diaminobenzidine was the most sensitive visualization method. It is easy to accomplish but a drawback is poor spatial resolution, which restricts its use at high magnifications. Nanogold visualization has considerably better spatial resolution and is therefore recommended for electron microscopy. Fluorescent tyramides, especially TRITC-tyramide, offer a good detection method for fluorescence and confocal microscopy. The methods were used to localize amphoterin and ␤ -actin mRNAs in motile cells. Both mRNAs were found in the soma and cell processes. In double labeling experiments, ␤ -actin mRNA localized to filamentous structures that also contained ribosomal proteins. Especially in the cortical cytoplasm, ␤ -actin mRNA was associated with actin filaments. Direct localization to microtubules was only rarely seen. S ubcellular compartmentalization of mRNA has been recognized as a mechanism for regulation of gene expression and protein sorting

Amphoterin, a major form of HMG (high mobility group) 1 proteins, is highly expressed in immature and malignant cells. A role in cell motility is suggested by the ability of amphoterin to promote neurite extension through RAGE (receptor of advanced glycation end products), an immunoglobulin superfamily member that communicates with the GTPases Cdc42 and Rac. We show here that cell contact with the laminin matrix induces accumulation of both amphoterin mRNA and protein close to the plasma membrane, which is accompanied by extracellular export of amphoterin. A role for amphoterin in extracellular matrix-dependent cell regulation is further suggested by the finding that specific decrease of amphoterin mRNA and protein, using antisense oligonucleotides transfected into cells, inhibits cell migration to laminin in a transfilter assay whereas the oligonucleotides in the culture medium have no effect. Moreover, affinity-purified anti-amphoterin antibodies inhibit cell migration to laminin, supporting an extracellular role for the endogenous amphoterin in cell motility. The finding that amphoterin expression is more pronounced in cells with a motile phenotype as compared to cells of dense cultures, is consistent with the results of the cell migration assays. Our results strongly suggest that amphoterin is a key player in the migration of immature and transformed cells.

Background: Humanized mice are a promising translational research model for evaluation of pharmacological compounds efficacy and safety in Oncology. Their use has been enhanced by the development of new stocks of immunodeficient hosts, most notably mouse strain such as NOD-scid/IL2rγ null mice (NSG). As previously described (L. Schultz et al, J. Immunol. 2005) the NSG mice could also be successfully humanized after engraftment of human hematopoietic stem cells (HSC). NSG mice have also been shown to be superior to other immunodeficient mice (BALB/c nu/nu, NOD/Shi-scid) for xenograft of tumour material. These models are particularly needed in preclinical development where there is no appropriate small animal model combining the human immune system and human tumour. Methods: Freshly collected umbilical cord blood (UCB) samples were provided by the “établissement français du sang, EFS” following the French ethical rules. The UCB were selected for their HSC CD34+ content and T cells depleted before being intracardiacally injected in whole body irradiated newborn NSG mice. The reconstitution of a human immune system within mouse was analyzed in peripheral blood and in primary and secondary lymphoid organs (bone marrow, spleen and thymus) using a 7-color FACS analysis. The mice were then xenografted with tumour cell lines, fresh primary human solid tumours and haematological malignancies to combine the development of human cancer and human immune system within the same mouse. Results and conclusions: Fifteen weeks after stem cells injection, the peripheral blood contains about 50% of human leukocytes distributed as followed: about 50% of T lymphocytes, about 40% of B lymphocytes, about few percent of monocytes, macrophages, granulocytes, natural killer cells and platelets. At same time, the central chimerism within lymphoid organs for human leucocytes has represented about 80%, 85% and 95% in bone marrow, spleen and thymus cells, respectively. This NSG mouse model opens new in vivo perspectives to study the complex relationships between the human immune system and human tumour cells under therapeutic treatments with biologics (antibody-dependent cell-mediated cytotoxicity, antitumour vaccination strategies . . .). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 394.

Localization of mRNAs is currently thought to be partially responsible for molecular sorting to specific compartments within the cell. In mammalian cells the best-studied example is the β-actin mRNA that is localized to the cell processes, and its localization is necessary in migratory responses of cells. It is reasonable to assume that mRNA localization within cells is coupled to transmembrane signalling due to extracellular factors, but little is known about such putative mechanisms. We show here that HB- GAM, an extracellular matrix-associated factor that enhances migratory responses in cells, is able to localize β- actin mRNA when locally applied to cells via microbeads. The HB-GAM-induced mRNA localization is specifically inhibited by low concentrations of heparin and by heparitinase treatment of cells, showing that cell-surface heparin-type glycans are required for the effect. The finding that soluble N-syndecan is also inhibitory suggests that the transmembrane proteoglycan N-syndecan, previously identified as an HB-GAM receptor, is involved in the mRNA-localizing effect of HB-GAM. Inhibition of the mRNA localization by the src-kinase inhibitor PP1 is compatible with an N-syndecan-mediated effect since the receptor function of N-syndecan has been recently found to depend on the src-kinase signalling pathway. The mRNA- localizing activity of N-syndecan is also suggested by the finding that affinity-purified anti-N-syndecan antibodies coated on microbeads are able to localize β-actin mRNA.

No abstract