ABSTRACT
Megakaryocytic differentiation of myelogenous leukemia cell lines induced by a number of chemical compounds mimics, in part, the physiological process that takes place in the bone marrow in response to a variety of stimuli. We have investigated the involvement of mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated protein kinase (ERK1/2) and p38] and phosphoinositide 3-kinase (PI3K) signaling pathways in the differentiated phenotypes of K562 cells promoted by phorbol 12-myristate 13-acetate, staurosporine (STA), and the p38 MAPK inhibitor SB202190. In our experimental conditions, only STA-treated cells showed the phenotype of mature megakaryocytes (MKs) including GPIbalpha expression, DNA endoreduplication, and formation of platelet-like structures. We provide evidence supporting that basal activity, but not sustained activation, of ERK1/2 is required for expression of MK surface markers. Moreover, ERK1/2 signaling is not involved in cell endomitosis. The PI3K pathway exerts dual regulatory effects on K562 cell differentiation: it is intimately connected with ERK1/2 cascade to stimulate expression of surface markers and it is also necessary, but not sufficient, for polyploidization. Finally, apoptosis and megakaryocytic differentiation exhibit different sensitivity to p38 down-regulation: it is required for expression of early specific markers but is not involved in cell apoptosis. The present work with K562 cells provides new insights into the molecular mechanisms regulating MK differentiation. The results indicate that a precise orchestration of signals, including ERK1/2 and p38 MAPKs as well as PI3K pathway, is necessary for acquisition of features of mature MKs.
Subject(s)
Cell Differentiation/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Megakaryocytes/cytology , Phosphatidylinositol 3-Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/physiology , Flow Cytometry , Humans , Imidazoles/pharmacology , K562 Cells , Megakaryocytes/drug effects , Phosphatidylinositol 3-Kinases/physiology , Phosphoinositide-3 Kinase Inhibitors , Polyploidy , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Signal Transduction , Tetradecanoylphorbol Acetate/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/physiologyABSTRACT
BACKGROUND AND OBJECTIVES: Glanzmann thrombasthenia is an autosomal recessive bleeding disorder characterized by a life-long hemorrhagic tendency and absent or severely reduced platelet aggregation in response to agonists, caused by quantitative or qualitative abnormalities in the platelet fibrinogen receptor, integrin alphaIIb beta3. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a patient with type II Glanzmann thrombasthenia. DESIGN AND METHODS: The expression of platelet alphaIIb beta3 was determined by flow cytometry and western blotting. Mutations were identified by sequencing both cDNA and genomic DNA. Functional characterization was assessed by exontrap and transient transfection analysis. RESULTS: Flow cytometry and western blot analysis revealed markedly reduced levels of platelet alphaIIb beta3, which may account for the residual fibrinogen binding detected upon platelet activation. Sequencing of genomic DNA revealed the presence of two mutations in the alphaIIb gene: a C1750T transition in the last codon of exon 17 changing Arg553 to STOP, and a C2829T transition in exon 27 that changes Pro912 to Leu. Sequence analysis of reversely transcribed alphaIIb mRNA did not detect cDNA from the C1750T mutant allele, and revealed a significant increase of the physiological splicing out of exon 28 in the cDNA carrying the C2829T mutation. Transient expression of [912Leu]alphaIIb in CHO-b3 cells showed a marked reduction in the rate of surface expression of alphaIIb beta3. INTERPRETATION AND CONCLUSIONS: The results suggest that the thrombasthenic phenotype is the result of reduced availability of alphaIIb-mRNA, enhanced expression of exon 28-deleted transcripts, and defective processing of [912Leu]alphaIIb.
Subject(s)
Exons/genetics , Genetic Carrier Screening , Mutation, Missense , Platelet Membrane Glycoprotein IIb/genetics , Thrombasthenia/genetics , Animals , CHO Cells , Child, Preschool , Cricetinae , Female , Humans , Thrombasthenia/classificationABSTRACT
We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.
