5 research outputs found

    Polarity of constitutive and regulated von Willebrand factor secretion by transfected MSDCK-II cells

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    Von Willebrand factor (vWF), synthesized by endothelial cells, is both rapidly secreted by the constitutive pathway and stored in Weibel-Palade bodies. Secretion from these organelles occurs upon activation of the protein kinase C signal transduction pathway and yields highly multimerized vWF. Highly multimerized vWF acts as a more effective adhesive ligand than the lower molecular weight forms that are constitutively secreted. We employed the extensively characterized polar Madin-Darby Canine Kidney II (MDCK-II) epithelial cell line, stably transfected with full-length vWF cDNA or deletion mutants thereof, to gain insight in the polarity of vWF secretion by either one of the two pathways. Immunofluorescence analysis and metabolic labeling experiments revealed that multimeric "wild-type" vWF is stored in MDCK-II cells and released upon stimulation with phorbol esters. Furthermore, we show that 62.0 +/- 3.8% of constitutively secreted and 83.2 +/- 6.6% of the regulated secreted wild-type vWF is encountered at the apical side of the cell. The polarity of the constitutive secretion of deletion mutant vWFdelD'D3 is similar to that of constitutively secreted wild-type vWF, whereas deletion mutant vWFdelD1D2 displays no polar secretion (50.1 +/- 5.7% apical

    Lack of gradual regulation of tetracycline-controlled gene expression by the tetracyclin-repressor/VP16 transactivator (tTA) in cultured cells

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    AbstractVon Willebrand factor (vWF) is an essential multimeric protein for adhesion of platelets to an injured vessel wall. Endothelial cells secrete vWF by either a constitutive or a regulated pathway. It is unknown whether the secretory partitioning of vWF is dependent on the level of vWF synthesis. We employed the widely applied tetracycline-controlled transactivator system (tTA) to study the regulation of vWF mRNA synthesis in stably transfected Madin Darby kidney (MDCK-II) cells in a quantitative manner. Immunofluorescence staining with anti-vWF antibodies revealed that increasing the concentration of tetracycline resulted in a decreased number of MDCK-II cells that synthesize vWF. Apparently, tTA-regulated gene expression in an individual cell functions as an ‘on/off’ system rather than regulating the level of gene expression in a dose-response manner, as reported previously.©1997 Federation of European Biochemical Societies
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