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M Pochet

Publications and source records attributed to M Pochet.

4 recordsLinked to original sources
PubMed2001

The role of CD47 in neutrophil transmigration. Increased rate of migration correlates with increased cell surface expression of CD47.

CD47, a cell surface glycoprotein, plays an important role in modulating neutrophil (PMN) migration across endothelial and epithelial monolayers. Here we show that anti-CD47 monoclonal antibodies (mAbs) delay PMN migration across collagen-coated filters or T84 epithelial monolayers toward the chemoattractant formylmethionylleucylphenylalanine (fMLP). Despite delayed transmigration by anti-CD47 mAbs, the numbers of PMN migrating across in either condition were the same as in the presence of control non-inhibitory mAbs. Cell surface labeling and immunoprecipitation demonstrated upregulation of CD47 to the PMN cell surface with kinetics similar to those of the transmigration response. Subcellular fractionation studies revealed redistribution of CD47 from intracellular compartments that co-sediment with secondary granules to plasma membrane-containing fractions after fMLP stimulation. Experiments performed to investigate potential signaling pathways revealed that inhibition of tyrosine phosphorylation with genistein reversed the anti-CD47-mediated PMN migration delay, whereas inhibition of phosphatidylinositol 3-kinase only partially reversed anti-CD47 effects that correlated with a rapid increase in PMN cell surface CD47. Analysis of the contribution of epithelial-expressed CD47 to PMN transmigration revealed that PMN migration across CD47-deficient epithelial monolayers (CaCO2) was significantly increased after stable transfection with CD47. These results suggest that cell surface CD47 and downstream tyrosine phosphorylation signaling events regulate, in part, the rate of PMN migration during the inflammatory response.

Antibodies, Monoclonal↗
PubMed2000

Human junction adhesion molecule regulates tight junction resealing in epithelia.

Epithelial cells form a highly selective barrier and line many organs. The epithelial barrier is maintained by closely apposed cell-cell contacts containing tight junctions, the regulation of which is incompletely understood. Here we report the cloning, tissue localization and evidence for a role in epithelial barrier regulation of an immunoglobulin superfamily member that likely represents the human homolog of murine junction adhesion molecule (JAM). Analysis of the primary structure of human JAM, cloned from T84 epithelial cells, predicts a transmembrane protein with an extracellular domain that contains two IgV loops. Monoclonal antibodies generated against the putative extracellular domain were reactive with a 35-39 kDa protein from both T84 epithelial cells and human neutrophils. By immunofluorescence, JAM mAbs labeled epithelial cells from intestine, lung, and kidney, prominently in the region of tight junctions (co-localization with occludin) and also along lateral cell membranes below the tight junctions. Flow cytometric studies confirmed predominant JAM expression in epithelial cells but also revealed expression on endothelial and hematopoietic cells of all lineages. Functional studies demonstrated that JAM specific mAbs markedly inhibited transepithelial resistance recovery of T84 monolayers after disruption of intercellular junctions (including tight junctions) by transient calcium depletion. Morphologic analysis revealed that, after disassembly of cell-cell junctions, anti-JAM inhibition of barrier function recovery correlated with a loss of both occludin and JAM, but not ZO-1, in reassembling tight junction structure. Reassembly of the major adherens junction component E-cadherin was not affected by JAM specific mAbs. Our findings suggest that JAM plays an important role in the regulation of tight junction assembly in epithelia. Furthermore, these JAM-mediated effects may occur by either direct, or indirect interactions with occludin.

Amino Acid Sequence↗
PubMed1998

Characterization of fatty acid synthase in cell lines derived from experimental mammary tumors.

The lipogenic enzyme fatty acid synthase (FAS) is elevated in various human primary cancers and certain human cancer cell lines. FAS overexpression in human neoplasia has clinical relevance because of its association with tumor aggression and potential chemotherapeutic intervention. Here, we surveyed FAS in cell lines established from normal murine mammary epithelium (NMuMG) and from mammary tumors induced by either rodent polyoma (Py) virus or murine mammary tumor virus (MMTV). Western blotting revealed greater content of FAS in Py-transformed A1-1 and T1 than NMuMG or MMTV-transformed Mm5MT, RIIIMT and MMT060562. These data suggest that signaling events mediated by Py transformation may increase cellular amounts of FAS. Although FAS content was elevated to similar levels in A1-1 and T1, specific activities were significantly different as enzyme activity in T1 was 3-fold higher than A1-1. Likewise, FAS activity in NMuMG was about 0.5-fold higher than the MMTV-transformed lines, even though enzyme content was similar. Immunoprecipitation studies employing anti-phosphoamino acid antibodies followed by immunoblot analysis with anti-FAS antisera (and vice versa) were used to characterize the constitutive phosphorylation state of the enzyme. Phosphoserine and phosphothreonine residues were detected in the more active FAS from T1 and NMuMG, but not in the less active FAS from Mm5MT or A1-1. Discovery of phosphorylated FAS suggests that the enzyme may have more immediate control over lipogenesis than previously thought. High-dose (10-4 M) dexamethasone induced FAS content and activity in NMuMG and MMTV-transformed lines but not Py-transformed cells. Lower concentrations (10-8, 10-6 M) of dexamethasone also activated FAS but without concomitant elevation of its protein content, which was consistent with a phosphorylated form of FAS. Finally, cell lines were treated with the FAS inhibitor cerulenin: almost all breast cancer lines were growth inhibited at significantly lower amounts of drug than normal cell lineages, suggesting that FAS plays a greater role in viability of tumor cells than normal cells. Pretreatment with palmitate (a primary end-product of FAS) prior to cerulenin rescued A1-1 cells only slightly from growth inhibition, whereas pretreatment with oleate (a monounsaturated fatty acid synthesized from palmitate) synergized cerulenin's cytotoxic effects.

Animals↗