cicaprost and Leukemia--Erythroblastic--Acute

We have previously demonstrated that activation of prostacyclin (IP) receptors in human erythroleukemia (HEL) cells phosphorylates the signal transducer and activator of transcription 3 (STAT3) via Gα(s) and Gα(16) hybrid signalling. This current study was designed to determine if functional responses to cicaprost in HEL cells were dependent on STAT3 phosphorylation. Cicaprost significantly enhanced the rapid change in HEL cell morphology induced by phorbol-12-myristate-13-acetate (PMA), and this effect was inhibited by the IP receptor antagonist RO1138452 and a STAT3 inhibitory peptide. Other indicators of PMA-induced HEL cell differentiation, such as increased expression of CD41/CD61 and an increase in cell complexity/granularity, were inhibited by cicaprost in an IP receptor-dependent and STAT3-dependent manner. Although thrombopoietic cytokines promote megakaryocytic differentiation and platelet production via activation of STAT3, the predominant STAT3-dependent effects of cicaprost in HEL cells were inhibitory towards the process of PMA-induced megakaryocytopoeisis.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Survival; Epoprostenol; Humans; Integrin beta3; Leukemia, Erythroblastic, Acute; Platelet Membrane Glycoprotein IIb; Receptors, Epoprostenol; Signal Transduction; STAT3 Transcription Factor; Tetradecanoylphorbol Acetate

Human prostacyclin receptor (hIP) stimulates STAT3 via pertussis toxin-insensitive G proteins in human erythroleukemia (HEL) cells. Since hIP can utilize G(s) and G(q) proteins for signal transduction and that both G proteins can induce STAT3 phosphorylation and activation via complex signaling networks, we sought to determine if one of them is predominant in mediating the hIP signal. Stimulation of STAT3 Tyr(705) and Ser(727) phosphorylations by the IP-specific agonist, cicaprost, was sensitive to inhibition of protein kinase A, phospholipase Cbeta, protein kinase C, calmodulin-dependent protein kinase II and Janus kinase 2/3. Unlike Galpha(16)-mediated regulation of STAT3 in the same cells, cicaprost-induced STAT3 Tyr(705) phosphorylation was resistant to inhibition of Src and MEK while STAT3 Ser(727) phosphorylation distinctly required phosphatidylinositol-3 kinase. This unique inhibitor-sensitivity pattern of STAT3 phosphorylation was reproduced in HEL cells by stimulating the G(16)-coupled C5a receptor in the presence of dibutyryl-cAMP, suggesting that the change in inhibitor-sensitivity was due to activation of the G(s) pathway. This postulation was confirmed by expressing constitutively active Galpha(16)QL and Galpha(s)QL in human embryonic kidney 293 cells and the inhibitor-sensitivity of Galpha(16)QL-induced STAT3 phosphorylations could be converted by the mere presence of Galpha(s)QL to resemble that obtained with cicaprost in HEL cells. In addition, the restoration of the Galpha(16)-mediated inhibitor-sensitivity upon cicaprost induction in Galpha(s)-knocked down HEL cells again verified the pivotal role of G(s) signal. Taken together, our observations illustrate that co-stimulation of G(s) and G(q) can result in the fine-tuning of STAT3 activation status, and this may provide the basis for cell type-specific responses following activation of hIP.

Topics: Adenylyl Cyclases; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; Epoprostenol; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Protein alpha Subunits, Gs; Humans; Janus Kinases; Leukemia, Erythroblastic, Acute; Luciferases; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotyrosine; Protein Kinase Inhibitors; Receptors, Epoprostenol; Signal Transduction; src-Family Kinases; STAT3 Transcription Factor

The ability of the human prostacyclin receptor (hIP) to regulate the activities of signal transducers and activators of transcription (STATs) has not yet been documented. In the present study, we have delineated the mechanism by which hIP induces STAT3 phosphorylations in human erythroleukemia (HEL) cells. Stimulation of endogenous hIP by its specific agonist, cicaprost, resulted in STAT3 Tyr705 and Ser727 phosphorylations in a time- and concentration-dependent manner. Cicaprost-induced STAT3 Tyr705 and Ser727 phosphorylations were resistant to pertussis toxin (PTX) treatment, suggesting that these responses were mediated through PTX-insensitive G proteins. In addition, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not p38 MAPK, were shown to be phosphorylated by cicaprost in a time- and concentration-dependent manner via PTX-insensitive G proteins. The levels of the interaction between STAT3, ERK and JNK were enhanced by cicaprost treatment. The involvement of Raf-1, MEK1/2 and JNK in cicaprost-induced phosphorylations of STAT3 was illustrated by the use of their selective inhibitors. In contrast, p38 MAPK did not appear to be required. Similar observations were obtained with STAT1 upon stimulation by cicaprost. Taken together, these results demonstrate for the first time that hIP activation by cicaprost can lead to STAT1 and STAT3 phosphorylations via signaling pathways involving PTX-insensitive G proteins, ERK and JNK.

Topics: Cell Line, Tumor; Dose-Response Relationship, Drug; Epoprostenol; Extracellular Signal-Regulated MAP Kinases; GTP-Binding Proteins; Humans; Leukemia, Erythroblastic, Acute; MAP Kinase Kinase 1; MAP Kinase Kinase 4; MAP Kinase Kinase Kinases; Pertussis Toxin; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Receptors, Epoprostenol; Serine; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Time Factors; Transcription, Genetic; Tyrosine

Like Ras, farnesylation of the IP (prostacyclin receptor) is required for its efficient intracellular signalling, and hence the IP represents a potential target for inhibition by FTIs [FTase (farnesyl protein transferase) inhibitors]. Herein, the effect of SCH66336 on the isoprenylation and function of the human and mouse IPs overexpressed in human embryonic kidney 293 cells, and by the IP endogenously expressed in human erythroleukaemia cells, was investigated. SCH66336 yielded concentration-dependent decreases in IP-mediated cAMP generation (IC50 0.27-0.62 nM), [Ca2+]i mobilization (IC50 26.6-48.3 nM) and IP internalization, but had no effect on signalling by the non-isoprenylated beta2 adrenergic receptor or b isoform of the TP (prostanoid thromboxane A2 receptor). Additionally, SCH66336 impaired IP-mediated crossdesensitization of TPa signalling (IC50 56.1 nM) and reduced farnesylation of the molecular chaperone protein HDJ-2 (IC50 3.1 nM). To establish whether farnesylation of the IP is inhibited and/or whether its 'CaaX motif' might undergo alternative geranylgeranylation in the presence of SCH66336, a series of chimaeric Ha (Harvey)-Ras fusions were generated by replacing its CaaX motif (-CVLS) with that of the IP (-CSLC) or, as controls, of Ki (Kirsten)-Ras 4B (-CVIM) or Rac 1 (-CVLL). Whereas SCH66336 had no effect on Ha-RasCVLL isoprenylation in vitro or in whole cells, it supported alternative geranylgeranylation of Ha-RasCVIM, but completely impaired isoprenylation of both Ha-RasCVLS and Ha-RasCSLC. These data confirm that the -CSLC motif of the IP is a direct target for inhibition by the FTI SCH66336, and in the presence of strong FTase inhibition, the IP does not undergo compensatory geranylgeranylation

Topics: Adrenergic beta-Agonists; Alkyl and Aryl Transferases; Amino Acid Motifs; Animals; Calcium Signaling; Carrier Proteins; Cell Line; Cell Line, Tumor; Cyclic AMP; Dose-Response Relationship, Drug; Endocytosis; Epoprostenol; Farnesyltranstransferase; Heat-Shock Proteins; HSP40 Heat-Shock Proteins; Humans; Iloprost; Isoproterenol; Kidney; Leukemia, Erythroblastic, Acute; Mice; Mutagenesis, Site-Directed; Organophosphorus Compounds; Piperidines; Proline; Propanolamines; Protein Prenylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); Pyridines; Receptors, Adrenergic, beta-2; Receptors, Epoprostenol; Receptors, Thromboxane A2, Prostaglandin H2; Recombinant Fusion Proteins; Signal Transduction; Transfection

The group of prostaglandin (PG) E2- and prostacyclin receptors consists of different subtypes, which exhibit different affinities for prostaglandins and synthetic analogues. PGE2 activities the E-type PG receptor subtypes EP1, EP2 and EP3, whereas the PGE2 analogue, sulprostone, binds only to the EP1 and EP3 receptor subtypes. The stable PGI2 analogues, iloprost and cicaprost, both activate the PGI2 receptor (IP) and iloprost, additionally, bind to the EP1 subtype. Using these subtype-selective PG receptor agonists, we studied the interaction of PG receptor subtypes with Gs and Gi-type heterotrimeric guanine nucleotide-binding proteins (G proteins) in membranes from the human erythroleukaemia cell line, HEL. Sulprostone stimulated high-affinity GTPase in HEL membranes in a pertussis toxin (PTX)-sensitive manner. In contrast, the stimulations induced by PGE2, iloprost and cicaprost were only partially inhibited by PTX. PGE2, sulprostone, iloprost and cicaprost stimulated cholera toxin-catalysed ADP-ribosylation as well as labelling with GTP azidoanilide of membrane proteins comigrating with immunologically identified Gi protein alpha subunits. Furthermore, PGE2, iloprost and cicaprost enhanced GTP azidoanilide-labelling of Gs protein alpha subunits, whereas sulprostone did not. We suggest that in HEL cells (1) EP1 and EP3 receptor subtypes activate G1 proteins, that (2) the EP2 receptor subtype activates Gs proteins and that (3) the IP receptor activates both Gi and Gs proteins.

Topics: Cell Membrane; Dinoprostone; Epoprostenol; GTP-Binding Proteins; Humans; Iloprost; Leukemia, Erythroblastic, Acute; Prostaglandins E; Receptors, Epoprostenol; Receptors, Prostaglandin; Receptors, Prostaglandin E; Signal Transduction; Tumor Cells, Cultured

In the human erythroleukemia cell line, HEL, prostaglandin E2 (PGE2) and the stable prostacyclin analogue, iloprost, increase cytosolic Ca2+ concentration ([Ca2+]i) via pertussis toxin-sensitive and -insensitive pathways. Unlike iloprost, the stable prostacyclin analogue cicaprost (ZK 96480), is devoid of agonistic properties at prostaglandin E2 receptors. We compared the effects of cicaprost, iloprost and PGE2 on [Ca2+]i in HEL cells. Cicaprost, iloprost and PGE2 were similarly potent to increase [Ca2+]i in HEL cells. However, unlike the effects of PGE2, those of the prostacyclin analogues were not inhibited by pertussis toxin. The prostaglandins studied increased [Ca2+]i through both mobilization from internal stores and Ca2+ influx from the extracellular space. Prostacyclin analogue- and PGE2-induced rises in [Ca2+]i were desensitized in a homologous manner. Additionally, there was cross-desensitization between cicaprost and iloprost, but not between the prostacyclin analogues and PGE2. Our data suggest that in HEL cells (i) cicaprost and iloprost act through prostacyclin receptors and (ii) that these receptors couple to pertussis toxin-insensitive heterotrimeric regulatory guanine nucleotide-binding proteins, (iii) resulting in an increase in [Ca2+]i by Ca2+ mobilization from internal stores and sustained influx.

Topics: Calcium; Cytosol; Dinoprostone; Epoprostenol; GTP-Binding Proteins; Humans; Iloprost; Kinetics; Leukemia, Erythroblastic, Acute; Pertussis Toxin; Tumor Cells, Cultured; Virulence Factors, Bordetella