Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells

The bisindolylmaleimide, GF109203X (2-[1-(3-dimethylaminopropyl)-1H- indol-3-yl]-3-(1H-indol-3-yl)-maleimide ), a highly selective inhibitor of protein kinase C (PKC), was used to test the role of this enzyme in phorbol ester-induced megakaryocytic differentiation of HEL cells. Treatment of these cells with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 3 days caused a complete inhibition of proliferation and a threefold increase in the surface expression of glycoprotein (GP) IIIa, a marker of megakaryocytic differentiation that forms part of the fibrinogen receptor complex, GPIIb/IIIa. A similar effect was observed with phorbol 12,13-dibutyrate, but not with the biologically inactive derivative PMA-4-O-methyl ether. The PMA-induced increase in GPIIIa expression was completely inhibited by GF109203X in a dose-dependent manner (IC50 = 0.5 mumol/L), with a maximal effect at 2.5 to 5.0 mumol/L. GF109203X also blocked the inhibitory effect of PMA on cell growth and inhibited PMA-stimulated phosphorylation of the 47-kD PKC substrate, pleckstrin. Incubation of HEL cells with 25 mumol/L hemin for 3 days caused a fourfold to fivefold increase in expression of the erythroid differentiation marker, glycophorin A. In contrast to the inhibitory effect of GF109203X on GPIIIa expression, hemin induction of glycophorin A was enhanced by this compound. Furthermore, GF109203X alone caused a dose-dependent increase in glycophorin A expression, and induced hemoglobinization. Consistent with these changes, Northern blot analysis revealed that GF109203X treatment reduced the steady-state level of GPIIb mRNA and increased those for glycophorin A and gamma-globin. These results suggest that PKC may act as a developmental switch controlling erythroid/megakaryocytic differentiation.

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Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells

Blood ◽

10.1182/blood.v87.1.123.123 ◽

1996 ◽

Vol 87 (1) ◽

pp. 123-131 ◽

Cited By ~ 50

Author(s):

Y Hong ◽

JF Martin ◽

W Vainchenker ◽

JD Erusalimsky

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Erythroid Differentiation ◽

Inhibitory Effect ◽

Glycophorin A ◽

Maximal Effect ◽

Megakaryocytic Differentiation ◽

Kinase C ◽

Hel Cells ◽

Dose Dependent

Abstract The bisindolylmaleimide, GF109203X (2-[1-(3-dimethylaminopropyl)-1H- indol-3-yl]-3-(1H-indol-3-yl)-maleimide ), a highly selective inhibitor of protein kinase C (PKC), was used to test the role of this enzyme in phorbol ester-induced megakaryocytic differentiation of HEL cells. Treatment of these cells with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 3 days caused a complete inhibition of proliferation and a threefold increase in the surface expression of glycoprotein (GP) IIIa, a marker of megakaryocytic differentiation that forms part of the fibrinogen receptor complex, GPIIb/IIIa. A similar effect was observed with phorbol 12,13-dibutyrate, but not with the biologically inactive derivative PMA-4-O-methyl ether. The PMA-induced increase in GPIIIa expression was completely inhibited by GF109203X in a dose-dependent manner (IC50 = 0.5 mumol/L), with a maximal effect at 2.5 to 5.0 mumol/L. GF109203X also blocked the inhibitory effect of PMA on cell growth and inhibited PMA-stimulated phosphorylation of the 47-kD PKC substrate, pleckstrin. Incubation of HEL cells with 25 mumol/L hemin for 3 days caused a fourfold to fivefold increase in expression of the erythroid differentiation marker, glycophorin A. In contrast to the inhibitory effect of GF109203X on GPIIIa expression, hemin induction of glycophorin A was enhanced by this compound. Furthermore, GF109203X alone caused a dose-dependent increase in glycophorin A expression, and induced hemoglobinization. Consistent with these changes, Northern blot analysis revealed that GF109203X treatment reduced the steady-state level of GPIIb mRNA and increased those for glycophorin A and gamma-globin. These results suggest that PKC may act as a developmental switch controlling erythroid/megakaryocytic differentiation.

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Protein kinase C isotypes in human erythroleukemia cell proliferation and differentiation

Journal of Cell Science ◽

10.1242/jcs.101.3.671 ◽

1992 ◽

Vol 101 (3) ◽

pp. 671-679

Author(s):

B.A. Hocevar ◽

D.M. Morrow ◽

M.L. Tykocinski ◽

A.P. Fields

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cellular Proliferation ◽

Biological Effects ◽

Growth Arrest ◽

Tumor Promoter ◽

Induced Differentiation ◽

Megakaryocytic Differentiation ◽

Kinase C ◽

Dose Dependent

The human erythroleukemia (K562) cell line is induced to differentiate into megakaryocytic cells by treatment with the tumor promoter phorbol myristate acetate (PMA). PMA-induced differentiation is characterized by (1) almost complete cessation of cellular proliferation, (2) expression of the megakaryocytic cell surface marker glycoprotein IIb/IIIa (gpIIIa), (3) increased secretion of granulocyte/macrophage-colony stimulating factor (GM-CSF) and (4) increased secretion of interleukin-6 (IL-6). PMA-induced differentiation is dose-dependent with maximal activity seen at 10 nM PMA. In contrast, bryostatin (bryo), a structurally distinct protein kinase C (PKC) activator, fails to induce megakaryocytic differentiation or growth arrest at the concentrations tested (0.01-100 nM). Rather, bryo inhibits PMA-induced growth arrest and megakaryocytic differentiation in a dose-dependent fashion (full inhibition at 100 nM). The divergent biological effects of PMA and bryo correspond to the differential activation and translocation of PKC isotypes in K562 cells. PKC isotype analysis demonstrates that undifferentiated cells express both alpha and beta II PKC but no detectable beta I, gamma or epsilon PKC. Treatment of cells with either PMA or bryo leads to rapid translocation of both alpha and beta II PKC from the cytosol to the non-nuclear particulate fraction. However, bryo also induces selective translocation of beta II PKC to the nuclear membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

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PMA‐induced megakaryocytic differentiation of HEL cells is accompanied by striking modifications of protein kinase C catalytic activity and isoform composition at the nuclear level

British Journal of Haematology ◽

10.1046/j.1365-2141.1996.00384.x ◽

1996 ◽

Vol 92 (3) ◽

pp. 530-536 ◽

Cited By ~ 28

Author(s):

Giorgio Zauli ◽

Alessandra Bassini ◽

Lucia Catani ◽

Davide Gibellini ◽

Claudio Celeghini ◽

...

Keyword(s):

Protein Kinase C ◽

Catalytic Activity ◽

Protein Kinase ◽

Nuclear Level ◽

Megakaryocytic Differentiation ◽

Kinase C ◽

Hel Cells

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Constitutive c-myb expression in K562 cells inhibits induced erythroid differentiation but not tetradecanoyl phorbol acetate-induced megakaryocytic differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.772 ◽

1995 ◽

Vol 15 (2) ◽

pp. 772-779 ◽

Cited By ~ 24

Author(s):

D Rosson ◽

T G O'Brien

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cellular Differentiation ◽

Plasmid Vector ◽

K562 Cells ◽

Erythroid Differentiation ◽

Tumor Promoter ◽

Megakaryocytic Differentiation ◽

Kinase C ◽

Tetradecanoyl Phorbol Acetate

K562 cells were stably transfected with a plasmid vector constitutively expressing a full-length human c-myb gene. Parental cells possess the dual potential of inducibility of cellular differentiation along two lineages, i.e., erythroid and megakaryocytic. The resulting lineage is dependent on the inducing agent, with a number of compounds being competent to various degrees for inducing erythroid differentiation, while the tumor promoter tetradecanoyl phorbol acetate (TPA) induces a macrophage-like morphology with enhanced expression of proteins associated with megakaryocytes. Exogeneous expression of c-myb in transfected cell lines abrogated erythroid differentiation induced by cadaverine or cytosine arabinoside as assessed by hemoglobin production. However, TPA-induced megakaryocytic differentiation was left intact, as assessed by cell morphology, cytochemical staining, and the expression of the megakaryocytic antigens. These results indicate that c-Myb and protein kinase C play important roles in cellular differentiation of K562 cells and suggest that agents which directly modulate protein kinase C can induce differentiation in spite of constitutively high levels of c-Myb.

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Osmotic Modulation of the Ouabain-Sensitive (Na++K+)ATPase from Malpighian Tubules of Rhodnius prolixus

Zeitschrift für Naturforschung C ◽

10.1515/znc-1998-9-1021 ◽

1998 ◽

Vol 53 (9-10) ◽

pp. 911-917 ◽

Cited By ~ 14

Author(s):

C. Caruso-Neves ◽

J. R. Meyer-Fernandes ◽

A. G. Lopes

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Atpase Activity ◽

Malpighian Tubule ◽

Inhibitory Effect ◽

Rhodnius Prolixus ◽

Malpighian Tubules ◽

Maximal Effect ◽

Kinase C ◽

Protein Kinase C Inhibitor

The presence and regulation by hyperosmotic medium of the ouabain-sensitive (Na++K+)ATPase of the Malpighian tubule cells of Rhodnius prolixus was investigated. The ouabain-sensitive (Na++K+)ATPase activity was 5.4 ± 0.5 nmol Pi x mg-1 x min-1. Vanadate 100 μM completely abolished this ATPase activity. In hyperosmotic medium, obtained by addition of 180 mᴍ mannitol, the (Na++K+)ATPase activity was inhibited by 60%. When the cell lysates were preincubated in hyperosmotic medium for 30 minutes and the ATPase activity was assayed in isosmotic medium, the (Na++K+)ATPase activity was not modified. Addition of 50 ng/ml sphingosine, a protein kinase C inhibitor, abolished the inhibition of (Na++K+)ATPase activity in hyperosmotic medium. Furtherm ore, phorbol ester (TPA), an activator of protein kinase C, mimicked the effect of hyperosmotic shock on (Na++K+)ATPase activity. The increase in Ca2+ concentration decreased the (Na++K+)ATPase activity by 60% in isosmotic medium, with maximal effect obtained in 10-6 m Ca2+. No effect was observed in hyperosmotic medium. The inhibitory effect of Ca2+ on the (Na++K+)ATPase was not reversed by sphingosine. These results indicate that the ouabain-sensitive (Na++K+) ATPase activity of the Malpighian tubule is regulated by both increasing Ca2+ concentration and by the osmolality of the medium by different and integrative ways.

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Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Cell Regulation ◽

10.1091/mbc.2.11.897 ◽

1991 ◽

Vol 2 (11) ◽

pp. 897-903 ◽

Cited By ~ 37

Author(s):

J K Pai ◽

E A Dobek ◽

W R Bishop

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase D ◽

Phorbol Esters ◽

Thymidine Incorporation ◽

Maximal Effect ◽

Kinase C ◽

Potent Vasoconstrictor ◽

Dose Dependent ◽

Protein Kinase C Beta

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.

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Identification and functional characterization of protein kinase C isozymes in platelets and HEL cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)50192-2 ◽

1992 ◽

Vol 267 (14) ◽

pp. 10011-10017

Author(s):

J Grabarek ◽

M Raychowdhury ◽

K Ravid ◽

K.C. Kent ◽

P.J. Newman ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Functional Characterization ◽

Kinase C ◽

Hel Cells

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Regulation of oxytocin secretion by the ovine corpus luteum: effect of activators of protein kinase C

Journal of Endocrinology ◽

10.1677/joe.0.1240225 ◽

1990 ◽

Vol 124 (2) ◽

pp. 225-232 ◽

Cited By ~ 1

Author(s):

J. J. Hirst ◽

G. E. Rice ◽

G. Jenkin ◽

G. D. Thorburn

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Corpus Luteum ◽

Phospholipase C ◽

Tissue Slices ◽

Kinase C ◽

Luteal Tissue ◽

Dose Dependent ◽

Stimulation Of

ABSTRACT The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234·4 ± 32·8 pmol/g per h (n = 24) during 60-min incubations.Activators of protein kinase C: phorbol 12,13-dibutyrate (n = 8), phorbol 12-myristate,13-acetate (n = 4) and 1,2-didecanoylglycerol (n = 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0·2 μmol/l). Phospholipase C (PLC; 50–250 units/l) also caused a dose-dependent stimulation of oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n = 4). These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum. Journal of Endocrinology (1990) 124, 225–232

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Prostacyclin inhibits platelet aggregation induced by phorbol ester or Ca2+ ionophore at steps distal to activation of protein kinase C and Ca2+-dependent protein kinases

Biochemical Journal ◽

10.1042/bj2580057 ◽

1989 ◽

Vol 258 (1) ◽

pp. 57-65 ◽

Cited By ~ 32

Author(s):

W Siess ◽

E G Lapetina

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Protein Kinases ◽

Shape Change ◽

Ionophore A23187 ◽

Maximal Effect ◽

Kinase C ◽

Dependent Protein ◽

High Concentrations

Suspensions of aspirin-treated, 32P-prelabelled, washed platelets containing ADP scavengers in the buffer were activated with either phorbol 12,13-dibutyrate (PdBu) or the Ca2+ ionophore A23187. High concentrations of PdBu (greater than or equal to 50 nM) induced platelet aggregation and the protein kinase C (PKC)-dependent phosphorylation of proteins with molecular masses of 20 (myosin light chain), 38 and 47 kDa. No increase in cytosolic Ca2+ was observed. Preincubation of platelets with prostacyclin (PGI2) stimulated the phosphorylation of a 50 kDa protein [EC50 (concn. giving half-maximal effect) 0.6 ng of PGI2/ml] and completely abolished platelet aggregation [ID50 (concn. giving 50% inhibition) 0.5 ng of PGI2/ml] induced by PdBu, but had no effect on phosphorylation of the 20, 38 and 47 kDa proteins elicited by PdBu. The Ca2+ ionophore A23187 induced shape change, aggregation, mobilization of Ca2+, rapid phosphorylation of the 20 and 47 kDa proteins and the formation of phosphatidic acid. Preincubation of platelets with PGI2 (500 ng/ml) inhibited platelet aggregation, but not shape change, Ca2+ mobilization or the phosphorylation of the 20 and 47 kDa proteins induced by Ca2+ ionophore A23187. The results indicate that PGI2, through activation of cyclic AMP-dependent kinases, inhibits platelet aggregation at steps distal to protein phosphorylation evoked by protein kinase C and Ca2+-dependent protein kinases.

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