Regulation of developing B cell survival by RelA-containing NF-kappa B complexes

Mice deficient in the RelA (p65) subunit of NF-kappaB die during embryonic development. Fetal liver (FL) hemopoietic precursors from these mice were used to generate RelA-deficient lymphocytes by adoptive transfer into lethally irradiated mature lymphocyte-deficient recombination-activating gene-1(-/-) mice. Strikingly, RelA(-/-) lymphocyte generation was greatly diminished compared with that of RelA(+/+) lymphocytes. The most dramatic reduction was noticed in the numbers of developing B cells, which were considerably increased when RelA(-/-) FL cells that were also TNFR1 deficient were used. The role of RelA was further investigated in FL-derived developing B cells in vitro. Our results show that RelA is a major component of constitutive and TNF-alpha-induced kappaB site-binding activity in developing B cells, and provide evidence for a direct role of TNF-alpha in killing RelA(-/-) B cells. The absence of RelA significantly reduced mRNA expression of the antiapoptotic genes cellular FLICE-inhibitory protein and Bcl-2. Retroviral transduction of RelA(-/-) B cells with either cFLIP or Bcl-2 significantly reduced TNF-alpha killing. Together, these results indicate that RelA plays a crucial role in regulating developing B cell survival by inhibiting TNF-alpha cytotoxicity.     

Regulation of layer-specific targeting by reciprocal expression of a cell adhesion molecule, capricious

Layer-specific innervation is a major form of synaptic targeting in the central nervous system. In the Drosophila visual system, photoreceptors R7 and R8 connect to targets in distinct layers of the medulla, a ganglion of the optic lobe. We show here that Capricious (CAPS), a transmembrane protein with leucine-rich repeats (LRRs), is a layer-specific cell adhesion molecule that regulates photoreceptor targeting in the medulla. During the period of photoreceptor targeting, caps is specifically expressed in R8 and its target layer but not in R7 or its recipient layer. caps loss-of-function mutations cause local targeting errors by R8 axons, including layer change. Conversely, ectopic expression of caps in R7 redirects R7 axons to terminate in the CAPS-positive R8 recipient layer. CAPS promotes homophilic cell adhesion in transfected S2 cells. These results suggest that CAPS regulates layer-specific targeting by mediating specific axon-target interaction.     

Regulation of endothelial cell adhesion molecule expression by mast cells, macrophages, and neutrophils

Background

Leukocyte adhesion to the vascular endothelium and subsequent transendothelial migration play essential roles in the pathogenesis of cardiovascular diseases such as atherosclerosis. The leukocyte adhesion is mediated by localized activation of the endothelium through the action of inflammatory cytokines. The exact proinflammatory factors, however, that activate the endothelium and their cellular sources remain incompletely defined.

Methods and Results

Using bone marrow-derived mast cells from wild-type, Tnf^−/−, Ifng^−/−, Il6^−/− mice, we demonstrated that all three of these pro-inflammatory cytokines from mast cells induced the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, and E-selectin in murine heart endothelial cells (MHEC) at both mRNA and protein levels. Compared with TNF-α and IL6, IFN-γ appeared weaker in the induction of the mRNA levels, but at protein levels, both IL6 and IFN-γ were weaker inducers than TNF-α. Under physiological shear flow conditions, mast cell-derived TNF-α and IL6 were more potent than IFN-γ in activating MHEC and in promoting neutrophil adhesion. Similar observations were made when neutrophils or macrophages were used. Neutrophils and macrophages produced the same sets of pro-inflammatory cytokines as did mast cells to induce MHEC adhesion molecule expression, with the exception that macrophage-derived IFN-γ showed negligible effect in inducing VCAM-1 expression in MHEC.

Conclusion

Mast cells, neutrophils, and macrophages release pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL6 that induce expression of adhesion molecules in endothelium and recruit of leukocytes, which is essential to the pathogenesis of vascular inflammatory diseases.     

Regulation of neural cell adhesion molecule expression on cultured mouse Schwann cells by nerve growth factor.

Schwann cells from early postnatal mouse sciatic nerve were obtained as a homogenous population and shown by indirect immunofluorescence to express the neural cell adhesion molecules L1, N-CAM and J1 and their common carbohydrate epitope L2/HNK-1. L1 and N-CAM are synthesized in molecular forms that are slightly different from those expressed by small cerebellar neurons or astrocytes. As in astrocytes, the J1 antigen is expressed by Schwann cells in multiple forms generally ranging from 160 to 230 kd in the reduced state. J1 is secreted by Schwann cells in a 230-kd mol. wt form. Expression of L1 by Schwann cells can be regulated by nerve growth factor (NGF). L1 expression on the cell surface is increased 1.6-fold in the presence of NGF after 3 days of maintenance in vitro and 3-fold after 16 days. NGF does not change expression of N-CAM. The glia-derived neurite-promoting factor (GdNPF) increases L1 expression by a factor of 1.9 and decreases N-CAM expression by a factor of 0.4 after 3 days in vitro. J1 expression on Schwann cell surfaces remains unchanged in the presence of NGF or GdNPF. Antibodies to NGF abolish the influence of NGF on L1 expression. Addition of NGF antibodies to the Schwann cell cultures without exogenously added NGF decreases L1 expression, indicating that Schwann cells secrete NGF that may influence L1 expression by an autocrine mechanism. Our experiments show for the first time that cell adhesion molecule expression on a non-neuronal cell, the Schwann cell, can be directly regulated by the neurotrophic factor NGF. These observations indicate a considerable degree of 'plasticity' of peripheral glia in regulating cell adhesion molecule expression.     

Regulation of human Ig lambda light chain gene expression by NF-kappa B.

The human Iglambda enhancer consists of three separated sequence elements that we identified previously by mapping DNase I-hypersensitive regions (HSS) downstream of the C region of the Iglambda L chain genes (HSS-1, HSS-2, and HSS-3). It has been shown by several laboratories that expression of the H chain genes as well as the kappa genes, but not the lambda genes, is dependent on constitutive NF-kappaB proteins present in the nucleus. In this study we show by band-shift experiments, in vivo footprinting, and transient transfection assays that all three hypersensitive sites of the human Iglambda enhancer contain functional NF-kappaB sites that act synergistically on expression. We further show that the chicken lambda enhancer also contains a functional NF-kappaB site but the mouse lambda enhancer contains a mutated, nonfunctional NF-kappaB site that is responsible for its low enhancer activity. It is possible that the inactivating mutation in the mouse Iglambda enhancer was compensated for by an expansion of the Igkappa L chain locus, followed by a contraction of the Iglambda locus in this species.     

Regulation of cell adhesion by polysialic acid. Effects on cadherin, immunoglobulin cell adhesion molecule, and integrin function and independence from neural cell adhesion molecule binding or signaling activity.

The polysialylation of neural cell adhesion molecule (NCAM) evolved in vertebrates to carry out biological functions related to changes in cell position and morphology. Many of these effects involve the attenuation of cell interactions that are not mediated through NCAM's own adhesion properties. A proposed mechanism for this global effect on cell interaction is the steric inhibition of membrane-membrane apposition based solely on polysialic acid (PSA) biophysical properties. However, it remains possible that the intrinsic binding or signaling properties of the NCAM polypeptide are also involved. To help resolve this issue, this study uses a quantitative cell detachment assay together with cells engineered to display different adhesion receptors together with a variety of polysialylated NCAM polypeptide isoforms and functional domain deletion mutations. The results obtained indicate that regulation by PSA occurs with adhesion receptors as diverse as an IgCAM, a cadherin and an integrin, and does not require NCAM functional domains other than those minimally required for polysialylation. These findings are most consistent with the cell apposition mechanism for PSA action, as this model predicts that the inhibitory effects of PSA-NCAM on cell adhesion should be independent of the nature of the adhesion system and of any intrinsic binding or signaling properties of the NCAM polypeptide itself.     

Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair

Peripheral nerve injury results in short-term and long-term changes in both neurons and glia. In the present study, immunohistological and immunoblot analyses were used to examine the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) within different parts of a functionally linked neuromuscular system extending from skeletal muscle to the spinal cord after peripheral nerve injury. Histological samples were taken from 3 to 150 d after crushing or transecting the sciatic nerve in adult chickens and mice. In unperturbed tissues, both N-CAM and Ng-CAM were found on nonmyelinated axons, and to a lesser extent on Schwann cells and myelinated axons. Only N-CAM was found on muscles. After denervation, the following changes were observed: The amount of N-CAM in muscle fibers increased transiently on the surface and in the cytoplasm, and in interstitial spaces between fibers. Restoration of normal N-CAM levels in muscle was dependent on reinnervation; in a chronically denervated state, N-CAM levels remained high. After crushing or cutting the nerve, the amount of both CAMs increased in the area surrounding the lesion, and the predominant form of N-CAM changed from a discrete Mr 140,000 component to the polydisperse high molecular weight embryonic form. Anti-N-CAM antibodies stained neurites, Schwann cells, and the perineurium of the regenerating sciatic nerve. Anti-Ng-CAM antibodies labeled neurites, Schwann cells and the endoneurial tubes in the distal stump. Changes in CAM distribution were observed in dorsal root ganglia and in the spinal cord only after the nerve was cut. The fibers within affected dorsal root ganglia were more intensely labeled for both CAMs, and the motor neurons in the ventral horn of the spinal cord of the affected segments were stained more intensely in a ring pattern by anti-N-CAM and anti-Ng-CAM than their counterparts on the side contralateral to the lesion. Taken together with the previous studies (Rieger, F., M. Grumet, and G. M. Edelman, J. Cell Biol. 101:285-293), these data suggest that local signals between neurons and glia may regulate CAM expression in the spinal cord and nerve during regeneration, and that activity may regulate N-CAM expression in muscle. Correlations of the present observations are made here with established events of nerve degeneration and suggest a number of roles for the CAMs in regenerative events.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of cell adhesion strength by peripheral focal adhesion distribution

Cell adhesion to extracellular matrices is a tightly regulated process that involves the complex interplay between biochemical and mechanical events at the cell-adhesive interface. Previous work established the spatiotemporal contributions of adhesive components to adhesion strength and identified a nonlinear dependence on cell spreading. This study was designed to investigate the regulation of cell-adhesion strength by the size and position of focal adhesions (FA). The cell-adhesive interface was engineered to direct FA assembly to the periphery of the cell-spreading area to delineate the cell-adhesive area from the cell-spreading area. It was observed that redistributing the same adhesive area over a larger cell-spreading area significantly enhanced cell-adhesion strength, but only up to a threshold area. Moreover, the size of the peripheral FAs, which was interpreted as an adhesive patch, did not directly govern the adhesion strength. Interestingly, this is in contrast to the previously reported functional role of FAs in regulating cellular traction where sizes of the peripheral FAs play a critical role. These findings demonstrate, to our knowledge for the first time, that two spatial regimes in cell-spreading area exist that uniquely govern the structure-function role of FAs in regulating cell-adhesion strength.     

Neural cell adhesion molecule expression is regulated by Schwann cell- neuron interactions in culture

To investigate the cellular and molecular signals underlying regulation of cell adhesion molecule expression, the influence of interactions between dorsal root ganglion neurons and Schwann cells on their expression of L1 and N-CAM was quantitated by immunogold electronmicroscopy. The numbers of antibody binding sites on cell surfaces of neurons and glia were compared between pure populations and co-cultures. After 3 d of co-culture, expression of L1 was reduced by 91% on Schwann cells and 36% on neurons, with expression in pure cultures being taken as 100%. N-CAM expression was unchanged on neurons and reduced by 43% on Schwann cells. Within 3 d after removal of neurons from Schwann cell-neuron co-cultures by immunocytolysis, expression of L1 and N-CAM on Schwann cell surfaces increased by 69 and 84%, respectively. Cell surface antigens recognized by an antibody to mouse liver membranes were unchanged in co-cultures. Furthermore, in co-cultures of neurons and sciatic nerve fibroblasts neither of the three antibodies detected any changes in expression of antigens when pure and co-cultures were compared. These observations suggest that adhesion molecules are not only involved in neuron-Schwann cell recognition and neurite outgrowth on Schwann cells (Seilheimer, B., and M. Schachner. 1988. J. Cell Biol. 107: 341-351), but that cell interactions, in turn, modulate the extent of adhesion molecule expression.     

Visualization of neural cell adhesion molecule by electron microscopy

The 130- and 160-kD polypeptide forms of the neural cell adhesion molecule (NCAM) were analyzed by electron microscopy after low angle rotary shadowing and freeze replication. Individual NCAM molecules appeared as uniformly thick rods, with a distinct bend or hinge region near their middle. Aggregates were also present, containing two to six rods in a pinwheel-like configuration without measurable overlap between rods. The 130- and 160-kD NCAM forms had lengths of 38 and 51 nm, respectively, with a difference in arm length distal to the bend, but not toward the center of the pinwheel. Although enzymatic removal of the polysialic acid moiety on NCAM did not alter the appearance of individual molecules, it did increase the average number of arms per aggregate. Monoclonal antibodies that recognize defined regions of the NCAM polypeptide were used to provide landmarks on the observed molecular figures. Two antibodies specific for cytoplasmic epitopes near the COOH terminus were clustered at the distal tip of aggregated arms. Two other antibodies that react with epitopes near the NH2 terminus and the middle of the molecule bound to sites more centrally located on the pinwheel structure. Together, these results suggest that the observed aggregates represent an association of molecules near their NH2-terminal homophilic binding site, and have led to several predictions about the nature of an NCAM-mediated cell-cell bond.     

Activated leukocyte cell adhesion molecule expression and shedding in thyroid tumors

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, cancers, and cancer-initiating cells. Alterations in expression of ALCAM have been reported in several human tumors, and cell adhesion functions have been proposed to explain its association with cancer. Here we documented high levels of ALCAM expression in human thyroid tumors and cell lines. Through proteomic characterization of ALCAM expression in the human papillary thyroid carcinoma cell line TPC-1, we identified the presence of a full-length membrane-associated isoform in cell lysate and of soluble ALCAM isoforms in conditioned medium. This finding is consistent with proteolytically shed ALCAM ectodomains. Nonspecific agents, such as phorbol myristate acetate (PMA) or ionomycin, provoked increased ectodomain shedding. Epidermal growth factor receptor stimulation also enhanced ALCAM secretion through an ADAM17/TACE-dependent pathway. ADAM17/TACE was expressed in the TPC-1 cell line, and ADAM17/TACE silencing by specific small interfering RNAs reduced ALCAM shedding. In addition, the CGS27023A inhibitor of ADAM17/TACE function reduced ALCAM release in a dose-dependent manner and inhibited cell migration in a wound-healing assay. We also provide evidence for the existence of novel O-glycosylated forms and of a novel 60-kDa soluble form of ALCAM, which is particularly abundant following cell stimulation by PMA. ALCAM expression in papillary and medullary thyroid cancer specimens and in the surrounding non-tumoral component was studied by western blot and immunohistochemistry, with results demonstrating that tumor cells overexpress ALCAM. These findings strongly suggest the possibility that ALCAM may have an important role in thyroid tumor biology.