title: Protein kinase C mediates the mitogenic action of thrombopoietin in c-Mpl-expressing UT-7 cells.
abstract: Protein kinase C (PKC) has been implicated in signal transduction events elicited by several hematopoietic growth factors. Thrombopoietin (TPO) is the major regulator of megakaryocytic lineage development, and its receptor, c-Mpl, transduces signals for the proliferation and differentiation of hematopoietic progenitors. In this study we have examined the effect of TPO on the subcellular distribution of PKC (a measure of enzyme activation) in a growth factor-dependent pluripotent hematopoietic cell line that was engineered to express the c-Mpl receptor (UT-7/mpl). In addition, we have assessed the significance of this activation for the induction of both mitogenesis and differentiation. Using a PKC translocation assay, TPO was found to stimulate a time- and dose-dependent increase in the total content of PKC activity present in the membrane fraction of UT-7/mpl cells (maximum increase = 2.3-fold above basal level after 15 minutes with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that TPO treatment led to a marked increase of the Ca2+/diacylglycerol-sensitive PKC isoforms alpha and beta found in the membrane fraction. In contrast, the subcellular distribution of these isoforms did not change after treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of UT-7/mpl cells to the selective PKC inhibitor GF109203X completely inhibited the PKC activity associated to the membrane fraction after TPO treatment, and blocked the mitogenic effect of TPO. In contrast, GF109203X had no effect on the TPO-induced expression of GpIIb, a megakaryocytic differentiation antigen. Downregulation of PKC isoforms alpha and beta to less than 25% of their initial level by treatment with phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic response, but had no significant effect when this response was induced by GM-CSF. Taken together, these findings suggest that (1) TPO stimulates the activation of PKC, (2) PKC activation mediates the mitogenic action of TPO, and (3) PKC activation is not required for TPO-induced expression of megakaryocytic surface markers.
authors.affiliation: Cruciform Project, Rayne Institute, University College London, UK.
authors.forename: Y
authors.initials: Y
authors.lastname: Hong
authors.forename: D
authors.initials: D
authors.lastname: Dumènil
authors.forename: B
authors.initials: B
authors.lastname: van der Loo
authors.forename: F
authors.initials: F
authors.lastname: Goncalves
authors.forename: W
authors.initials: W
authors.lastname: Vainchenker
authors.forename: J D
authors.initials: JD
authors.lastname: Erusalimsky
mesh_terms: D001692:Biological Transport; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D008533:Megakaryocytes / Q000166:cytology; D008934:Mitogens / Q000494:pharmacology*; D009363:Neoplasm Proteins; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D015398:Signal Transduction; D013926:Thrombopoietin / Q000494:pharmacology*
chemical_list: D004075:Diglycerides; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D008934:Mitogens; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D013926:Thrombopoietin; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D002118:Calcium