Regulation of tie receptor expression on human endothelial cells by protein kinase C-mediated release of soluble tie

• Published in: Blood Vol. 90; no. 2; pp. 706 - 715
• Main Authors: YABKOWITZ, R, MEYER, S, YANAGIHARA, D, BRANKOW, D, STALEY, T, ELLIOTT, G, HU, S, RATZKIN, B
• Format: Journal Article
• Language: English
• Published: Washington, DC The Americain Society of Hematology 15.07.1997
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Calcium - pharmacology; CHO Cells; Cricetinae; Edetic Acid - pharmacology; Egtazic Acid - pharmacology; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Enzyme Activation; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glycosylation; Humans; Kinetics; Molecular Weight; Oligosaccharides - analysis; Protease Inhibitors - pharmacology; Protein Kinase C - metabolism; Receptor Protein-Tyrosine Kinases - biosynthesis; Receptor Protein-Tyrosine Kinases - chemistry; Receptor Protein-Tyrosine Kinases - isolation & purification; Receptors, TIE; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Sialic Acids - analysis; Tetradecanoylphorbol Acetate - pharmacology; Transfection; Transferases; Umbilical Veins; Human; Cell culture; Endothelial cell; Molecular form; Enzyme; Transferases; Soluble form; Membrane receptor; Regulation(control); Animal; Established cell line; Protein-tyrosine kinase; Biological receptor
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V90.2.706

The expression and activity of receptor tyrosine kinases (RTK) at the cell surface can be modulated by several different pathways including the proteolytic release of the extracellular domain as a soluble receptor. We investigated the regulation of tie receptor expression, an orphan RTK restricted to cells of hematopoietic and endothelial lineages, on primary human endothelial cells and a stably transfected Chinese hamster ovary (CHO) cell line. Tie was expressed in cells as a doublet of 135 and 125 kD; the 135-kD band represented mature cell surface receptor containing sialic acid and N-linked oligosaccharide residues, whereas the 125-kD band represented an intracellular pool of immature receptor. Phorbol 12-myristate 13-acetate (PMA) had dramatic effects on tie expression at the cell surface. Within 15 minutes of PMA treatment, the 135-kD band disappeared from the cell surface and was accompanied by the appearance of a 100-kD band in cell supernatants. The 100-kD band continued to accumulate in the media throughout the duration of PMA treatment during which mature tie receptor was undetectable on the cell surface by fluorescence-activated cell sorting (FACS) or in cell lysates by immunoblot analysis. Using specific antibodies, this 100-kD species was shown to be a soluble form of the tie receptor containing the extracellular domain. PMA-dependent release of soluble tie was mediated through the activation of protein kinase C (PKC); soluble tie was not released in the presence of PKC inhibitors, an inactive PMA analog, or following the downregulation of PKC through chronic PMA treatment. These results indicate that tie receptor expression on endothelial cells is regulated by the release of a soluble extracellular fragment following activation of PKC. Parallel pathways regulating c-kit, tumor necrosis factor (TNF), and colony-stimulating factor (CSF) receptor expression suggest that the release of extracellular receptor fragments represents an alternative mechanism through which cells modulate responses to growth factors and cytokines.