Hyaluronan-oligosaccharide-induced transcription of metalloproteases.

Activated dendritic epidermal Langerhans cells and metastatic tumour cells share many properties. Both cell types can invade the surrounding tissue, enter the lymphatic system and travel to regional lymph nodes. We have recently shown that fragments of the extracellular matrix component hyaluronan, which are typically produced at sites of inflammation, can activate dendritic cells. Upon activation, dendritic cells upregulate expression of matrix metalloproteases (MMPs). These observations prompted us to investigate whether exposure to hyaluronan fragments also induces MMP expression in tumour cells. Here, we report that MMP-9, MMP-13 and urokinase plasminogen activator are upregulated in murine 3LL tumour cells after exposure to mixed-size hyaluronan. Similarly upregulated MMP-9 and MMP-13 expression was observed in primary fibroblasts. By using size-fractionated hyaluronan preparations, we show that the enhanced expression of MMP-9 and MMP-13 is only induced by small hyaluronan (HA) fragments. Although our data suggest that HA-fragment-induced MMP-9 and MMP-13 expression is receptor mediated, they rule out an involvement of the hyaluronan receptors CD44, RHAMM/IHAP and TLR-4. Finally, we show that HA fragment-induced MMP-9 transcription is mediated via NF-kappa B. Our results suggest that the metastasis-associated HA degradation in tumours might promote invasion by inducing MMP expression.

Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions.

Nogo-A is a potent neurite growth inhibitor in vitro and plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-terminus stain the cell surface of living cultured oligodendrocytes. Nogo-A is also labeled by nonmembrane-permeable biotin derivatives applied to living oligodendrocyte cultures. Immunofluorescent staining of intracellular, endoplasmic reticulum-associated Nogo-A in cells after selective permeabilization of the plasma membrane reveals that the epitopes of Nogo-A, shown to be accessible at the cell surface, are exposed to the cytoplasm. This suggests that Nogo-A could have a second membrane topology. The two proposed topological variants may have different intracellular as well as extracellular functions.

Rapid expression cloning of receptors using epitope-tagged ligands and high-speed cell sorting.

BACKGROUND: In this study we describe a new approach for expression cloning of receptors. METHODS: Our approach was based on highly efficient transfer of retroviral cDNA libraries into target cells and detection of receptor-ligand interaction with the use of an antibody directed against an epitope tag on recombinant ligands. Detection of the complex and isolation of receptor-transduced cells were achieved by flow cytometry and rare event high-speed cell sorting. Recovery of the cDNA coding for the receptor(s) was achieved by polymerase chain reaction. RESULTS: As a proof-of-concept study we set out to clone the receptor for B-lymphocyte stimulator protein (BlyS), not known at the start of the project but reported while this work was in progress. First, we detected binding of epitope-tagged BlyS to IM9 cells. Second, human T-lymphoblasts (CEM cells), which do not bind BlyS, were transduced with a retroviral cDNA library generated from IM9 cells. Transduced CEM cells binding epitope-tagged BlyS protein were identified by flow cytometry. After three sequential rounds of cell sorting, transduced CEM cell populations with high binding capacity for BlyS were identified. To determine the cDNAs conferring binding to the transduced CEM cells, the integrated proviral DNAs were amplified by polymerase chain reaction and analyzed by DNA sequencing. Rescued cDNAs contained Transmembrane Activator and calcium-modulator and cyclophilin ligand (CAML) Interactor (TACI) and B-Cell Maturation factor (BCMA) sequences, representing two published receptors of BlyS. CONCLUSIONS: Our data demonstrated that flow cytometry and high-speed cell sorting combined with transduction of retroviral cDNA libraries and binding of epitope-tagged orphan ligands as a selectable phenotype can be used efficiently for expression cloning of receptors. Of particular interest was our finding that apparently it is not necessary to purify the ligand but that conditioned medium containing the ligand can be used instead. Thus we concluded that our approach shortens the time to identify receptors for many orphan ligands and helps to exploit these receptors as drug targets.