Regulation of annexin I in rheumatoid synovial cells by glucocorticoids and interleukin-1

The glucocorticoid (GC)-induced antiinflammatory molecule annexin I is expressed in leukocytes and has antiinflammatory effects in animal models of arthritis, but the expression of annexin I in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) is unknown. We report the constitutive and dexamethasone (DEX)-inducible expression of annexin I in RA FLS. DEX increased FLS annexin I protein translocation and mRNA expression. Interleukin (IL)-1beta also induced annexin I translocation and mRNA but also increased intracellular protein. DEX and IL-1 had additive effects on annexin I mRNA, but DEX inhibited the inducing effect of IL-1beta on cell surface annexin I. These results indicate that glucocorticoids and IL-1beta upregulate the synthesis and translocation of annexin I in RA FLS, but interdependent signalling pathways are involved.     

In vivo growth conditions suppress the expression of ganglioside GM2 and favour that of lacto series gangliosides in the human glioma D-54MG cell line

The human glioma D-54MG cell line grownin vitro primarily expresses ganglio series gangliosides, particularly GM2. Subcutaneous injection of these cells into nude mice produced xenografts with an increased content of the human glioma-associated lacto series gangliosides, primarily 3-isoLM1, an alteration that was dose dependent, with the highest dose (1×10⁸) resulting in a phenotype that was most like that of the inoculum. After one passagein vivo, the lacto series dominated and reached a proportional level that was kept throughout the 10 passages. The mRNA levels of the GM2-synthase clearly coincided with GM2 expression and was 20 times higher in cells grownin vitro than in those grownin vivo. These results support the view that ganglioside expression in human gliomas is strongly influenced by environmental factors. Abbreviations: The gangliosides have been designated according to Svennerholm (Eur J Biochem (1977)79: 11–21) GM3, II³NeuAc-LacCer; GM2, II³NeuAc-GgOse₃Cer; GM1, II³NeuAc-GgOse₄Cer; GD3, II³(NeuAc)₂-LacCer; GD2, II³(NeuAc)₂-GgOse₃Cer; GD1a, IV³NeuAc, II³NeuAc-GgOse₄Cer; GD1b, II³(NeuAc)₂-GgOse₄Cer; GT1b, IV³NeuAc, II³(NeuAc)₂-GgOse₄Cer; 3-LM1, IV³NeuAc-nLcOse₄Cer; 3-isoLM1, IV³NeuAc-LcOse₄Cer; 3,6-isoLD1, IV³NeuAc, III⁶NeuAc-LcOse₄Cer; 38-LM1, IV³(NeuAc)₂-nLcOse₄Cer. MAb(s), monoclonal antibody (ies); the designation LM1 is used when both 3-isoLM1 and 3-LM1 and LD1, when both 36-isoLD1 and 38-LD1 are included.     

Accumulation of sulfatide in neuronal and glial cells of arylsulfatase A deficient mice

Arylsulfatase A (ASA) degrades sulfatide, seminolipid and lactosylceramide sulfate, glycolipids recognized by the Sulph I antibody although sulfatide is considered the main antigen. Sulfatide is myelin associated but studies have shown a minor distribution also in non-myelin forming cells. The aim of this work was to further study sulfatide in neurons and astrocytes by immunohistochemistry, facilitated by investigation of tissue from adult ASA deficient (ASA ?/?) mice. Cells with a low presence of sulfatide might be detected due to lack of ASA activity and accumulation of Sulph I antigens. Sulfatide positive astrocytes and neurons were more numerous and intensely stained in ASA ?/? mice, demonstrating a sulfatide accumulation compared to controls. Sulph I staining was especially increased in the molecular layer of cerebellum, in which Purkinje cell dendrites displayed an altered morphology, and in layer IV–VI of cerebral cortex. In hippocampus, immunostaining was found in neuronal cytoplasm in ASA ?/? but in nuclear membranes of control mice. We observed a gray matter astrogliosis, which appeared to be associated to sulfatide accumulation. In addition, the developmental change (<20 months) of Sulph I antigens, galactosylceramide, phospholipids and cholesterol were followed by lipid analyses which verified sulfatide and seminolipid accumulation in adult ASA ?/? mice, although no lactosylceramide sulfate could be detected. In addition to demonstrating sulfatide in neurons and astrocytes, this study supports the value of ASA ?/? mice as a model for metachromatic leukodystrophy and suggests that accumulation of sulfatide beyond myelin might contribute to the pathology of this disease.     

Role of the proteasome in modulating native G-CSFR expression

The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis, but the mechanisms controlling its surface expression are poorly understood. Recent studies using transfected cell lines have suggested the activated G-CSFR is routed to the lysosome and not the proteasome. Here, we examined the role of the ubiquitin/proteasome system in regulating G-CSFR surface expression in both ts20 cells that have a temperature-sensitive E1 ubiquitin-activating enzyme and in primary human neutrophils. We show that the G-CSFR is constitutively ubiquitinated, which increases following ligand binding. In the absence of a functional E1 enzyme, ligand-induced internalization of the receptor is inhibited. Pre-treatment of ts20 transfectants with either chloroquine or MG132 inhibited ligand-induced G-CSFR degradation, suggesting a role for both lysosomes and proteasomes in regulating G-CSFR surface expression in this cell line. In neutrophils, inhibition of the proteasome but not the lysosome was found to inhibit internalization/degradation of the activated G-CSFR. Collectively, these data demonstrate the requirement for a functional ubiquitin/proteasome system in G-CSFR internalization and degradation. Our results suggest a prominent role for the proteasome in physiologic modulation of the G-CSFR, and provide further evidence for the importance of the ubiquitin/proteasome system in the initiation of negative signaling by cytokine receptors.     

Central role of the exchange factor GEF-H1 in TNF-α–induced sequential activation of Rac,ADAM17/TACE,and RhoA in tubular epithelial cells

Transactivation of the epidermal growth factor receptor (EGFR) by tumor necrosis factor-α (TNF-α) is a key step in mediating RhoA activation and cytoskeleton and junction remodeling in the tubular epithelium. In this study we explore the mechanisms underlying TNF-α–induced EGFR activation. We show that TNF-α stimulates the TNF-α convertase enzyme (TACE/a disintegrin and metalloproteinase-17), leading to activation of the EGFR/ERK pathway. TACE activation requires the mitogen-activated protein kinase p38, which is activated through the small GTPase Rac. TNF-α stimulates both Rac and RhoA through the guanine nucleotide exchange factor (GEF)-H1 but by different mechanisms. EGFR- and ERK-dependent phosphorylation at the T678 site of GEF-H1 is a prerequisite for RhoA activation only, whereas both Rac and RhoA activation require GEF-H1 phosphorylation on S885. Of interest, GEF-H1-mediated Rac activation is upstream from the TACE/EGFR/ERK pathway and regulates T678 phosphorylation. We also show that TNF-α enhances epithelial wound healing through TACE, ERK, and GEF-H1. Taken together, our findings can explain the mechanisms leading to hierarchical activation of Rac and RhoA by TNF-α through a single GEF. This mechanism could coordinate GEF functions and fine-tune Rac and RhoA activation in epithelial cells, thereby promoting complex functions such as sheet migration.     

Monoclonal anti-GD3 antibodies selectively inhibit the proliferation of human malignant glioma cells in vitro

The frequently occurring alteration of ganglioside expression in tumor cells has been implicated to play a role in the uncontrolled growth of these cells; antibodies to such gangliosides might affect tumor cell growth. We have studied the effect of IgM monoclonal antibodies to two glioma-associated gangliosides, GD3 and GM2, on cell proliferation of four human glioma cell lines and one renal tumor cell line. Of the two anti-ganglioside antibodies tested, only the anti-GD3 antibody resulted in a significant (p<0.005) inhibition of cell proliferation as measured by thymidine incorporation and Brd-U labeling, after 24[emsp4 ]h incubation. The effect was not dependent on any serum factor and no increased cell death was observed. All cell lines contained higher or similar amounts of GM2 than GD3, and both antigens were shown to be expressed on the cell surface and accessible to antibodies. The selective effect of anti-GD3 antibodies as contrasted to the inactivity of anti-GM2 antibodies suggests a possible role for ganglioside GD3 in tumor cell proliferation.     

Immune response to a synthetic peptide corresponding to an epitope of a parasitophorous vacuole membrane antigen from Plasmodium falciparum

The parasitophorous vacuole membrane antigen QF 116 from Plasmodium falciparum contains a defined epitope, DNNLVSGP, proximal to the carboxyl-terminus which binds to the inhibitory monoclonal antibody 8E7/55. A synthetic peptide containing this epitope was constructed and coupled to diphtheria toxoid as carrier. Mice and rabbits were inoculated with this conjugate using CFA, SAF-1, or aluminum phosphate as adjuvants. The peptide conjugate was highly immunogenic in both animal species, giving rise to polyclonal antibodies with a similar epitope specificity as the original mAb. Antibody titers were dependent on the route of immunization. Rabbit antibodies produced in sufficient quantity for biologic assays inhibited parasite growth in vitro. This synthetic peptide thus shows promise as an immunogen for use in synthetic vaccine design.