Emerin prevents BAF-mediated aggregation of lamin A on chromosomes in telophase to allow nuclear membrane expansion and nuclear lamina formation.

Several studies have suggested a role for the LEM-domain protein emerin and the DNA binding factor BAF in nuclear envelope reformation after mitosis, but the exact molecular mechanisms are not understood. Using HeLa cells deficient for emerin or both emerin and lamin A, we show that emerin deficiency induces abnormal aggregation of lamin A at the nuclear periphery in telophase. As a result, nuclear membrane expansion is impaired and BAF accumulates at the core region, the middle part of telophase nuclei. Aggregates do not form when lamin A carries the mutation R435C in the immunoglobulin fold known to prevent interaction of lamin A with BAF suggesting that aggregation is caused by a stabilized association of lamin A with BAF bound to chromosomal DNA. Reintroduction of emerin in the cells prevents formation of lamin A clusters and BAF accumulation at the core region. Therefore emerin is required for the expansion of the nuclear membrane at the core region to enclose the nucleus and for the rapid reformation of the nuclear lamina based on lamin A/C in telophase. Finally, we show that LEM-domain and lumenal domain are required for the targeting of emerin to exert its function at the core region.

Association of stomatin with lipid-protein complexes in the plasma membrane and the endocytic compartment.

Membrane protein - microvilli - lipid raft - GPI-anchored protein - epithelial cell The 31 kDa integral membrane protein stomatin (protein 7.2b) has a monotopic structure and a cytofacial orientation. We have shown previously that stomatin is located in plasma membrane protruding structures and forms high-order homo-oligomers in the human epithelial cell line UAC, suggesting that this protein has a structural function in the cortical morphogenesis of the cells. It is also present in a pool of juxtanuclear vesicles. In this study, we show that stomatin colocalizes with the GPI-anchored proteins placental alkaline phosphatase (PLAP) and membrane folate receptor alpha (MFRalpha) endogenously expressed in UAC cells. This observation enabled us to demonstrate two different aspects of stomatin. First, using anti-PLAP antibody internalization, we show that the peri-centrosomal vesicles containing stomatin correspond to a subset of endosomes, which can also be labeled with the late endosomal/lysosomal marker LAMP-2. Secondly, we found that stomatin is partially present in detergent-insoluble membrane domains and co-patches with PLAP on the plasma membrane, after cross-linking of PLAP by antibodies. These data indicate that stomatin and GPI-anchored proteins are linked through lipid rafts and undergo the same sorting events. We propose that stomatin, through its affinity for lipid rafts, functions in concentrating GPI-anchored proteins in membrane microvillar structures. Consistent with this hypothesis, we found that stomatin is expressed exclusively in microvilli of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.

Cysteine 29 is the major palmitoylation site on stomatin.

The 31 kDa membrane protein stomatin was metabolically labeled with tritiated palmitic acid in the human amniotic cell line UAC and immunoprecipitated. We show that the incorporated palmitate is sensitive to hydroxylamine, indicating the binding to cysteine residues. Stomatin contains three cysteines. By expressing a myc-tagged stomatin and substituting the three cysteines by serine, individually or in combination, we demonstrate that Cys-29 is the predominant site of palmitoylation and that Cys-86 accounts for the remaining palmitate labeling. Disruption of Cys-52 alone does not show any detectable reduction of palmitic acid incorporation. Given the organization of stomatin into homo-oligomers, the presence of multiple palmitate chains is likely to increase greatly the affinity of these oligomers for the membrane and perhaps particular lipid domains within it.

Oligomeric nature of the integral membrane protein stomatin.

The 31-kDa integral membrane protein stomatin (protein 7.2b) is not only an important component of the red cell membrane but can also be found in abundance in different tissues and cell lines. The protein is thought to be anchored to the membrane by a hydrophobic domain while both N and C termini are exposed to the cytoplasm. We have previously shown in the human cell line UAC that stomatin concentrates preferentially in plasma membrane folds and protrusions. There is also evidence that stomatin is linked to the cortical actin cytoskeleton, suggesting a role in cortical morphogenesis of the cell. In this study, we demonstrate that the fundamental structure of stomatin is oligomeric. Whereas interaction of stomatin with itself was suggested by cross-linking experiments, we show by density gradient centrifugation analysis that soluble homo-oligomeric complexes of this protein are present in Triton X-100 extracts of UAC cells. We also show the existence of these oligomers by co-immunoprecipitation of the endogenous stomatin and a recombinantly expressed myc-tagged stomatin, using an anti-myc antibody. The data indicate that these complexes comprise between 9 and 12 monomers of stomatin. Two C-terminally truncated forms of stomatin do not incorporate into these oligomers, suggesting an involvement of the C terminus in the homo-oligomeric interaction.

Colocalization of stomatin (band 7.2b) and actin microfilaments in UAC epithelial cells.

Cytolocalization of stomatin, an integral membrane protein also called erythrocyte band 7.2b, was investigated in a human epithelial cell line in which the expression of this protein is up-regulated after treatment with interleukin-6 and dexamethasone. A monoclonal antibody against stomatin was used to perform immunofluorescence and immunoelectron microscopy. The data show that stomatin concentrates preferentially in small plasma membrane protrusions. It is also found in abundance in a juxtanuclear structure possibly derived from the Golgi apparatus. Fluorescent double staining using the anti-stomatin antibody and the actin binding drug phalloidin shows a significant degree of colocalization of stomatin and cortical actin microfilaments. This association remains after actin filament disruption disruption by cytochalasin D treatment indicating a strong connection between stomatin and the membrane-associated cytoskeleton.

Cloning and chromosomal localization of a pseudogene corresponding to a mRNA for a soluble IL-6 receptor.

A polymerase chain reaction assay (Lust J.A. et al. (1992). Cytokine 4:96-100) was used to detect a mRNA coding for a soluble IL-6 receptor in human hepatoma cells. In addition to the expected amplification product, we found a sequence (SR4) which could be aligned to it with 78% identity. After cloning and sequencing a genomic 2.5-2.7 kB EcoRI fragment containing SR4, this sequence turned out to be part of a pseudogene corresponding to the transmembrane domain deleted soluble IL-6 receptor. Screening of a panel of interspecies hybrids revealed that it maps to chromosome 9.

Induction of metallothionein and stomatin by interleukin-6 and glucocorticoids in a human amniotic cell line.

Interleukin 6 (IL-6) is an important mediator of various kinds of inflammatory and immune responses. The human amniotic cell line UAC has an increased number of IL-6 receptors after treatment by glucocorticoids. To find a possible activity of IL-6 on these cells, a cDNA library of IL-6- and dexamethasone-treated cells was screened with cDNA probes from both induced and non-induced cells. Two cDNAs showed a differential hybridization signal. The first one corresponds to metallothionein, a group of small cysteine-rich proteins thought to participate in the metabolism and storage of zinc and to protect cells against oxidative damage. A second cDNA corresponds to the recently cloned cDNA of band 7 integral membrane protein also called stomatin. In hereditary stomatocytosis, absence of this protein in erythrocyte membranes is associated with high Na+ and low K+ intracellular concentrations [Stewart, G. W., Hepworth-Jones, B. E., Keen, J. N., Dash, B. C. J., Argent, A. C. & Casimir, C. M. (1992) Blood 79, 1593-1601]. In UAC cells both metallothionein and stomatin are induced by dexamethasone and IL-6 in a more than additive manner. Western blot analysis shows that stomatin protein is induced in a similar way as its mRNA. IL-6 and dexamethasone induce a state of resistance against hydrogen peroxide toxicity in UAC cells. Metallothionein induction might be partly responsible for this cytoprotection against oxidative stress.

Enhancement of IL-6 receptor beta chain (gp130) expression by IL-6, IL-1 and TNF in human epithelial cells.

Analysis of the IL-6 Receptor beta chain (gp130) mRNA expression on the two human epithelial cell lines UAC and Hep3B reveals that it is enhanced by IL-6, IL-1 and TNF treatment. In the case of UAC cells, TNF action might be mediated by IL-6. For Hep3B cells, TNF seems to exert a direct effect on gp130, as no IL-6 expression is detected after stimulation by this cytokine. On the same cells, increase of the binding of an anti-gp130 monoclonal antibody was observed after treatment by TNF, which denotes the effective appearance of new gp130 molecules on the cell surface. All this cytokines seem to act selectively on the beta chain of the IL-6 receptor. This probably reflects the importance for some cells to have gp130 represented on their membrane in inflammatory contexts.

Fate of interleukin-6 in the rat. Involvement of skin in its catabolism.

Iodinated recombinant human interleukin-6 (125I-rhIL-6) was intravenously injected into rats and its fate was studied during 24 h. Between 10-20 min after a single-dose injection, 125I-rhIL-6 accumulated in liver as previously reported [Castell et al. (1988) Eur. J. Biochem. 177, 357-361]. After 1 h, the radioactivity disappeared from the liver and accumulated in skin, reaching 35% of injected 125I-rhIL-6 5-8 h after injection. No comparable accumulation of radioactivity was found in skin when [125I]iodide or rat serum 125I-albumin was administered. Finally the radioactivity was detected as [125I]iodide in urine. Autoradiographic analysis of skin sections 5 h after 125I-rhIL-6 injection showed radioactivity in the interstitium. When the experiments were carried out with [35S]rhIL-6, essentially the same results were obtained: a decrease in radioactivity in the liver after 20 min, and a substantial increase in skin 7 h after injection. In vitro experiments showed that 125I-rhIL-6 is degraded by rat and human fibroblasts, whereas no degradation was observed with rat hepatoma cells (Fao) or human hepatocytes. These observations suggest the involvement of skin in the catabolism of IL-6.

Glucocorticoid up-regulation of high-affinity interleukin 6 receptors on human epithelial cells.

Interleukin 6 (IL-6) is a potent pleiotropic cytokine, known, among others, to stimulate immunoglobulin production by B cells and to trigger acute-phase protein synthesis by hepatocytes. Similar to IL-1, it is produced by monocytes and macrophages following an inflammatory challenge. Analysis of IL-6 receptor (IL-6R) expression on different human cell lines indicates that dexamethasone could up-regulate the number of IL-6R on one epithelial cell line (UAC) and on two hepatoma cell lines (HepG2 and Hep3B). This effect was confirmed by Scatchard analysis of binding experiments, using [35S]methionine and [35S]cysteine metabolically labeled IL-6. It was confirmed at the level of mRNA expression by Northern blot analysis. These results provide evidence for a link between IL-6 and glucocorticoids. They could represent an example of a system in which one role of glucocorticoids is to define more accurately the target of cytokines, and they could explain, at least partly, the frequently observed synergy between IL-6 and glucocorticoids, notably in the case of hepatocytes.