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Identification of signalling cascades involved in red blood cell shrinkage and vesiculation.

Kostova EB, Beuger BM, Klei TR, Halonen P, Lieftink C, Beijersbergen R, van den Berg TK, van Bruggen R - Biosci. Rep. (2015)

Bottom Line: In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors.Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity.In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation.

View Article: PubMed Central - PubMed

Affiliation: *Department of Blood Cell Research, Sanquin Research, Plesmanlaan 125, 1066CX, Amsterdam, The Netherlands.

ABSTRACT
Even though red blood cell (RBC) vesiculation is a well-documented phenomenon, notably in the context of RBC aging and blood transfusion, the exact signalling pathways and kinases involved in this process remain largely unknown. We have established a screening method for RBC vesicle shedding using the Ca(2+) ionophore ionomycin which is a rapid and efficient method to promote vesiculation. In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors. We investigated compounds triggering vesiculation and compounds inhibiting vesiculation induced by ionomycin. We identified 12 LOPAC compounds, nine kinase inhibitors and one kinase activator which induced RBC shrinkage and vesiculation. Thus, we discovered several novel pathways involved in vesiculation including G protein-coupled receptor (GPCR) signalling, the phosphoinositide 3-kinase (PI3K)-Akt (protein kinase B) pathway, the Jak-STAT (Janus kinase-signal transducer and activator of transcription) pathway and the Raf-MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinase) pathway. Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity. In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation.

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Scatter plots and gating strategy of RBC with DMSO (A) and with (B) ionomycin stimulationIn a control setting with DMSO treatment alone (A) RBC demonstrate 2500 events in P1, 7500 events in P2 and 200 events in P3. Upon stimulation with 5 μM ionomycin at 37°C for 30 min (B), RBC shrink due to Ca2+ flux and K+ efflux, leading to vesiculation (arrow) accompanied by an increase in events in P2 to 9000. Plots represent one of six independent measurements.
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Figure 1: Scatter plots and gating strategy of RBC with DMSO (A) and with (B) ionomycin stimulationIn a control setting with DMSO treatment alone (A) RBC demonstrate 2500 events in P1, 7500 events in P2 and 200 events in P3. Upon stimulation with 5 μM ionomycin at 37°C for 30 min (B), RBC shrink due to Ca2+ flux and K+ efflux, leading to vesiculation (arrow) accompanied by an increase in events in P2 to 9000. Plots represent one of six independent measurements.

Mentions: First, we performed four primary screens: LOPAC inhibition and induction screens and Kinase Inhibitors Library inhibition and induction screens. Each LOPAC screen contained 1280 small molecules with known biological functions in the fields of cell signalling and neuroscience, including apoptosis, gene regulation and expression, lipid signalling, neurotransmission, phosphorylation, ion channel transport and G-protein signalling. The Selleckchem Kinase Inhibitor Library consisted of 192 inhibitors specifically targeting kinases from various families. We performed inhibition and induction screens in parallel since we aimed at identifying molecules that induce RBC vesiculation (induction screens) and molecules that inhibit vesiculation induced by ionomycin (inhibition screens). In a control setting without stimulation (DMSO alone), RBC scatter could be depicted by three populations (P) each containing a specific number of events. P1 contained 2500 events; P2 contained 7500 events, whereas P3, the gate in which larger vesicles/microparticles could be observed, contained 200 events (Figure 1A). When RBCs were treated with ionomycin, a change in scatter accompanied by a considerable change in the number of events in all the three gates was observed. Events from P1 moved to P2, which reached over 9000 events, corresponding to a reduction in side scatter (cell shrinkage); furthermore around 500 events were detected in P3 (Figure 1B; arrow). However, in our analysis, we refer to RBC vesiculation as increase in events in gate P2. We chose this parameter and not the increase in P3, as the majority of vesicles will be too small to be detected on the flow cytometer and other events, such as debris, also fall into this gate. This renders P3 unsuitable for quantification of the effects of the different compounds. On the other hand, cell shrinkage due to membrane loss invariably occurs following vesiculation and can be depicted as an increase in the number of events in P2, accompanied by a decrease in events in P1. As a calcium ionophore, ionomycin induces calcium influx in RBC, which stimulates the calcium-activated potassium channel KCNN4 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4) (IK-1, inwardly rectifying potassium current; SK4, small conductance calcium-activated potassium channel 4; Gardos channel) leading to potassium efflux, cell dehydration, cell shrinkage, PS exposure and ultimately vesicle release [31–33]. As inducer of vesiculation, ionomycin was used as a positive control in all induction screens. After analysis of the primary LOPAC screens we identified 123 compounds inducing vesiculation and 162 compounds inhibiting vesiculation, which accounted for roughly 10% of all tested compounds. However, of each set we selected the top 2.3% of all compounds, considering normal distribution, which accounted for the 29 hits that were chosen for further validation. After analysis of the primary kinase inhibitor screens, it became clear that the inhibition screen did not yield any hits. For the induction screen of the Kinase Inhibitor Library we selected hits for further validation based on z-score, which accounted for 21 compounds in total.


Identification of signalling cascades involved in red blood cell shrinkage and vesiculation.

Kostova EB, Beuger BM, Klei TR, Halonen P, Lieftink C, Beijersbergen R, van den Berg TK, van Bruggen R - Biosci. Rep. (2015)

Scatter plots and gating strategy of RBC with DMSO (A) and with (B) ionomycin stimulationIn a control setting with DMSO treatment alone (A) RBC demonstrate 2500 events in P1, 7500 events in P2 and 200 events in P3. Upon stimulation with 5 μM ionomycin at 37°C for 30 min (B), RBC shrink due to Ca2+ flux and K+ efflux, leading to vesiculation (arrow) accompanied by an increase in events in P2 to 9000. Plots represent one of six independent measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400636&req=5

Figure 1: Scatter plots and gating strategy of RBC with DMSO (A) and with (B) ionomycin stimulationIn a control setting with DMSO treatment alone (A) RBC demonstrate 2500 events in P1, 7500 events in P2 and 200 events in P3. Upon stimulation with 5 μM ionomycin at 37°C for 30 min (B), RBC shrink due to Ca2+ flux and K+ efflux, leading to vesiculation (arrow) accompanied by an increase in events in P2 to 9000. Plots represent one of six independent measurements.
Mentions: First, we performed four primary screens: LOPAC inhibition and induction screens and Kinase Inhibitors Library inhibition and induction screens. Each LOPAC screen contained 1280 small molecules with known biological functions in the fields of cell signalling and neuroscience, including apoptosis, gene regulation and expression, lipid signalling, neurotransmission, phosphorylation, ion channel transport and G-protein signalling. The Selleckchem Kinase Inhibitor Library consisted of 192 inhibitors specifically targeting kinases from various families. We performed inhibition and induction screens in parallel since we aimed at identifying molecules that induce RBC vesiculation (induction screens) and molecules that inhibit vesiculation induced by ionomycin (inhibition screens). In a control setting without stimulation (DMSO alone), RBC scatter could be depicted by three populations (P) each containing a specific number of events. P1 contained 2500 events; P2 contained 7500 events, whereas P3, the gate in which larger vesicles/microparticles could be observed, contained 200 events (Figure 1A). When RBCs were treated with ionomycin, a change in scatter accompanied by a considerable change in the number of events in all the three gates was observed. Events from P1 moved to P2, which reached over 9000 events, corresponding to a reduction in side scatter (cell shrinkage); furthermore around 500 events were detected in P3 (Figure 1B; arrow). However, in our analysis, we refer to RBC vesiculation as increase in events in gate P2. We chose this parameter and not the increase in P3, as the majority of vesicles will be too small to be detected on the flow cytometer and other events, such as debris, also fall into this gate. This renders P3 unsuitable for quantification of the effects of the different compounds. On the other hand, cell shrinkage due to membrane loss invariably occurs following vesiculation and can be depicted as an increase in the number of events in P2, accompanied by a decrease in events in P1. As a calcium ionophore, ionomycin induces calcium influx in RBC, which stimulates the calcium-activated potassium channel KCNN4 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4) (IK-1, inwardly rectifying potassium current; SK4, small conductance calcium-activated potassium channel 4; Gardos channel) leading to potassium efflux, cell dehydration, cell shrinkage, PS exposure and ultimately vesicle release [31–33]. As inducer of vesiculation, ionomycin was used as a positive control in all induction screens. After analysis of the primary LOPAC screens we identified 123 compounds inducing vesiculation and 162 compounds inhibiting vesiculation, which accounted for roughly 10% of all tested compounds. However, of each set we selected the top 2.3% of all compounds, considering normal distribution, which accounted for the 29 hits that were chosen for further validation. After analysis of the primary kinase inhibitor screens, it became clear that the inhibition screen did not yield any hits. For the induction screen of the Kinase Inhibitor Library we selected hits for further validation based on z-score, which accounted for 21 compounds in total.

Bottom Line: In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors.Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity.In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation.

View Article: PubMed Central - PubMed

Affiliation: *Department of Blood Cell Research, Sanquin Research, Plesmanlaan 125, 1066CX, Amsterdam, The Netherlands.

ABSTRACT
Even though red blood cell (RBC) vesiculation is a well-documented phenomenon, notably in the context of RBC aging and blood transfusion, the exact signalling pathways and kinases involved in this process remain largely unknown. We have established a screening method for RBC vesicle shedding using the Ca(2+) ionophore ionomycin which is a rapid and efficient method to promote vesiculation. In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors. We investigated compounds triggering vesiculation and compounds inhibiting vesiculation induced by ionomycin. We identified 12 LOPAC compounds, nine kinase inhibitors and one kinase activator which induced RBC shrinkage and vesiculation. Thus, we discovered several novel pathways involved in vesiculation including G protein-coupled receptor (GPCR) signalling, the phosphoinositide 3-kinase (PI3K)-Akt (protein kinase B) pathway, the Jak-STAT (Janus kinase-signal transducer and activator of transcription) pathway and the Raf-MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinase) pathway. Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity. In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation.

Show MeSH
Related in: MedlinePlus