Precise contact between epithelial cells and their underlying basement membrane is crucial to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG gene (Dag1) expression in cultured mammary epithelial cells. Strikingly, DG loss disrupted laminin-111-induced polarity and β-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. Dystroglycan re-expression restored these deficiencies. Investigations of the mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when the entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in mammary epithelial cells that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity and β-casein induction. The observed loss of laminin-111 assembly and signaling in Dag1-/- mammary epithelial cells provides insights into the signaling changes occurring in breast carcinomas and other cancers, where the binding function of DG to laminin is frequently defective.

We report the identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein (hVAP-33) that has been implicated in neuronal exocytosis in Aplysia californica. This hVAP-33 shared 50% amino acid identity with the A. californica form and had similar length, structural organization and VAMP-binding abilities. However, in contrast with the neuron-specific expression seen in A. californica, hVAP-33 was broadly expressed, suggesting possible roles in vesicle fusion in both neuronal and non-neuronal cells.

Interferon-alpha (IFN-alpha) (IFN-alpha2b) is an immunoregulatory cytokine that is presently used in a recombinant form for the treatment of tumours and chronic viral infection. However, its mechanism of action remains largely undefined. In this paper, we studied the effects of low doses of IFN-alpha (0-100 U/ml) on the generation of dendritic cells with granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumour necrosis factor (TNF)-alpha in cultures of human peripheral blood mononuclear cells (PBMCs). An addition of IFN-alpha to the PBMC cultures greatly increased the HLA class II and the CD86 expression on developing dendritic cells (DCs) during a 7-day culture period. When added at the initiation of the PBMC culture, as little as 10 U/ml dramatically increased the HLA class II and CD86 expression, with maximal effects observed between 50 and 100 U/ml in all PBMC preparations tested. Almost all of the nonadherent cells induced with added IFN-alpha possessed a phenotype of mature DCs, being CD1a(low), CD83+, HLA class IIhigh, CD86high, CD40high, and CD80low, while being negative for the monocyte/macrophage and lymphocyte markers. In contrast, the floating cells isolated from cultures grown without IFN-alpha were mostly immature DCs with a CD1a(high), CD83-, HLA class IIint/high, CD86low/int, CD80low phenotype. An addition of 50 U/ml IFN-alpha at the time of the culture initiation greatly increased both the number of mature DCs generated and their rate of appearance; by 3 days of culture, many large floating aggregates were present containing mature CD83+, CD1a(low) DCs, while much fewer aggregates of mature DCs were found without added IFN-alpha. Histochemical staining confirmed that the floating cells induced with IFN-alpha had typical DC features, including irregularly shaped nuclei, few cytoplasmic granules, and absent or diffuse perinuclear staining for esterase. Our results suggest that IFN-alpha is a potent accelerator of DC maturation in vitro. These effects on DC maturation may explain its clinical success in the treatment of cancer and viral infection as well as its ability to promote autoimmunity.

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Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are critical proteins in membrane fusion, in both regulated and constitutive vesicular traffic. In addition, proteins that interact with the SNAREs are thought to regulate fusion. Vesicle-associated membrane protein-2 (VAMP-2) is a SNARE protein involved in insulin-dependent glucose transporter 4 (GLUT4) traffic. VAMP-2 is required for productive GLUT4 incorporation into the plasma membrane. VAMPassociated protein of 33 kDa (VAP-33) is an integral membrane protein that binds VAMPs in vitro, and is hypothesized to be a regulator of VAMPs. In L6 skeletal myoblasts, which display insulin-dependent traffic of GLUT4, we show that VAP-33 colocalized significantly with VAMP-2 using indirect confocal immunofluorescence and biochemical cosegregation. Overexpression of wild-type VAP-33 in L6 myoblasts attenuated the insulindependent incorporation of myc-tagged GLUT4 into the plasma membrane, and this response was restored by co-overexpression of VAMP-2 linked to green fluorescent protein. Antibodies to VAP-33 microinjected into 3T3-L1 adipocytes abrogated the insulin-stimulated translocation of GLUT4 to the plasma membrane, as measured in adhered plasma membrane lawns. Immunopurified VAMP-2-containing compartments from L6 myotubes and 3T3-L1 adipocytes showed significant levels of VAP-33. We propose that VAP-33 may be a regulator of VAMP-2 availability for GLUT4 traffic and other vesicle fusion events. Key words: Endosome, exocytosis, SNARE, VAMP-2, vesicle traffic Received 24 January 2000, revised and accepted for publication 16 March 2000Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins have been the focus of multiple studies into vesicle-target membrane fusion. Syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) in one membrane and vesicle-associated membrane protein (VAMP) in an opposing membrane are thought to coalesce to form a higher order coiled-coil complex that mediates fusion (1). Primary, secondary, tertiary and quaternary structural features of SNAREs are conserved from yeast to mammals (2 -5) and the energy released during the formation of these quaternary coiled-coil structures has been proposed to contribute to vesicle fusion (1). Reconstitution experiments have demonstrated that SNAREs suffice for fusion in vitro (6). Multiple isoforms of each SNARE have been identified, each having a distinct tissue and subcellular distribution (for a comprehensive review of SNARE isoforms see (7)).In addition, specific proteins bind to individual SNARE molecules and are therefore considered to be potential SNARE regulators. Two of these SNARE-binding proteins, munc-18c and syntaxin interacting protein (synip), interact selectively with syntaxin-4 in 3T3-L1 adipocytes, and the extent of these interactions is regulated by insulin (8 -11). A protein interacting with the SNARE VAMP was also identified in Aplysia californica and called VAMP-associated protein of 33 kDa (VAP-33) (12). Antibodies to ...

We tested the hypothesis that osteopontin (OPN) can inhibit the induction of inducible nitric oxide synthase (iNOS) in vascular tissue. iNOS activity was induced in rat thoracic aortas by incubation of the tissue with lipopolysaccharide (LPS) and measured by conversion ofl-[3H]arginine tol-[3H]citrulline. Addition of ≥1 nM recombinant OPN protein significantly reduced the LPS-induced increase in iNOS activity. Western blotting and the RT-PCR were used to determine the effect of LPS with and without OPN on tissue levels of iNOS protein and RNA, respectively. LPS resulted in an increase in iNOS protein and RNA, whereas OPN dose-dependently reduced tissue levels of iNOS activity, protein, and RNA. Mutated OPN proteins, in which the integrin-binding RGD amino acid sequence was deleted or mutated to RGE, resulted in complete and partial loss, respectively, of the ability of OPN to inhibit LPS-induced iNOS activity, implicating integrin binding in the effect. These results indicate that OPN can prevent induction of iNOS in vascular tissue.

Dystroglycan (DG) is a single receptor that binds to multiple basement membrane proteins and forms a transmembrane link to the actin cytoskeleton. It was first isolated as a component of the dystrophin-glycoprotein complex, which plays a role in the maintenance of muscle cell integrity and is defective in many muscular dystrophies. Although studied most extensively in muscle tissues, DG is present at most cell-basement membrane interfaces, and only recently has investigation of DG functions in nonmuscle cells gained momentum. Information emerging from recent studies in epithelial cells is implicating DG in a wide range of critical cell responses to the basement membrane, ranging from organization of tissue architecture to cell survival. Moreover, DG functions appear to be frequently absent in carcinoma cells, implicating its loss in cancer progression. Although many questions remain as to its precise role in mammary tissue, DG is emerging as a potentially important player in mammary gland function.

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