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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 of RBC treated with LOPAC compounds palmitoyl-DL-carnitine chloride, calmidazolium chloride, SCH-202676 hydrobromide and rac-2-ethoxy-3-hexadecanamido-1-propylphosphocholinePalmitoyl-DL-carnitine chloride (PKC modulator; A) and calmidazolium chloride (CaM antagonist; B) induced vesiculation assessed by transfer of all events from P2 to P3. SCH-2022676 (GPCR modulator) caused vesiculation (increased number of events in P3) but no shrinkage (no change in P2; C), whereas rac-2-ethoxy-3-hexadecanamido-1-propylphosphocholine had a pronounced effect on RBC preventing the measurement of any events (D). RBCs were treated with the respective compounds for 30 min at 37°C, followed by flow cytometry. All compounds were diluted in DMSO and added at 10 μM final concentration. Plots represent one of three independent measurements.
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Figure 2: Scatter plots of RBC treated with LOPAC compounds palmitoyl-DL-carnitine chloride, calmidazolium chloride, SCH-202676 hydrobromide and rac-2-ethoxy-3-hexadecanamido-1-propylphosphocholinePalmitoyl-DL-carnitine chloride (PKC modulator; A) and calmidazolium chloride (CaM antagonist; B) induced vesiculation assessed by transfer of all events from P2 to P3. SCH-2022676 (GPCR modulator) caused vesiculation (increased number of events in P3) but no shrinkage (no change in P2; C), whereas rac-2-ethoxy-3-hexadecanamido-1-propylphosphocholine had a pronounced effect on RBC preventing the measurement of any events (D). RBCs were treated with the respective compounds for 30 min at 37°C, followed by flow cytometry. All compounds were diluted in DMSO and added at 10 μM final concentration. Plots represent one of three independent measurements.

Mentions: After analysis of the induction screens, we identified 12 compounds from LOPAC inducing cell shrinkage/vesiculation and 10 compounds from kinase inhibitor set inducing cell shrinkage/vesiculation. All validated hits from LOPAC and kinase inhibitor induction screens are listed in Tables 1 and 2 respectively. In case of the LOPAC screen, the description includes relevant effects of the compounds not only provided by Sigma (denoted by a star *) but also functions described in literature. Validated hits had a median of the replicates value (Rep median score) higher than 1, meaning they caused an increase in the number of events in P2 compared with controls. Nevertheless, four LOPAC compounds were identified by analysis of RBC scatter alone and not by score value for the following reasons: palmitoyl-DL-carnitine chloride and calmidazolium chloride induced massive vesiculation in RBC leading to a concentration of all events in P3 (Figures 2A and 2B), therefore an increase in P2 could not be used as a parameter. SCH-202676 hydrobromide, on the other hand, induced vesiculation measured by an increase in P3 but no shrinkage (Figure 2C), whereas rac-2-ethoxy-3-hexadecanamido-1-propylphoshocholine's effect on RBC scatter was so strong, no events could be measured (Figure 2D). The adjusted P-value (Padj score) of all hits in the induction screens was ≤0.1, which was considered significant, with the exception of bromoacetyl alprenolol menthane and NNC 55–0396 from the LOPAC (Table 1) and AT7867 and NVP-TAE684 from the Kinase Inhibitor Library (Table 2). Nevertheless, these compounds were included in the validated hit list as they all had Rep median score higher than 1 (Tables 1 and 2). In addition, these compounds induced RBC shrinkage/vesiculation (Figures 4H, 4I, 5J and 5K), measured by a decrease in RBC side scatter, indicating reduced cell size (Figures 4J and 5L). The compounds from the LOPAC library inducing cell shrinkage/vesiculation could be clustered into four functional groups: PKC activity (six compounds including ET-18-OCH3 [34], calcium signalling (two compounds), GPCR signalling (three compounds) and protease activity (one compound) [35] (Figure 3A). Overall these results underscored the potential of our screening method, since the effects of PKC signalling [28], Ca2+ [26] and calpain [27] on RBC have been well studied. In addition, we identified GPCR signalling for the first time to be involved in RBC vesiculation. Figures 2 and 4 illustrate the effects of all LOPAC hits on RBC scatter. Compared with RBC treated with DMSO alone (Figure 4A), it is clear that all validated hits induce RBC shrinkage, measured by a significant increase in the number of events in P2 (Figures 4B–4G), with the exception of bromoacetyl alprenolol menthane and NNC 55–0396 (Figures 4H and 4I), which induced RBC shrinkage measured by a decrease in RBC side scatter (Figure 4J). Since the Kinase Inhibitor Library consisted exclusively of specific kinase inhibitors, we clustered the validated hits according to the family of kinases they target (Figure 3B). Interestingly, we discovered not only compounds targeting kinases with known functions in RBC to induce vesiculation (Table 2), but also compounds inhibiting kinases were described to be expressed in RBC, such as Alk [36] and VEGFR-2 [37]. We could confirm VEGFR-2 was found in RBC with a Western blot (result not shown). Figure 5 shows representative scatter plots of RBC treated with all validated kinase inhibitors. All induction compounds caused a significant increase in P2 compared with treatment with DMSO alone (Figures 5B–5I), with the exception of NVP-TAE684 and AT7867 (Figures 5J and 5K), which induced shrinkage measured by a significant decrease in RBC side scatter (Figure 5L). In addition, NVP-TAE684 treatment lead to vesiculation, measured by an increase in events in P3 (Figure 5J). Moreover, we generated response plots of the hits identified in the kinase inhibitor induction validation screen demonstrating how strong the effect of each compound was (Supplementary Figure). Compound concentrations (100 nM, 1 μM and 1 0μM) were plotted on the x-axis whereas the normalized P2 value was plotted on the y-axis. The response to each compound was represented by a solid line. The closer the line was to the positive control (dotted red line), the stronger the effect was. No compounds inhibiting vesiculation upon ionomycin stimulation were identified during the LOPAC validation inhibition screen and the Kinase Inhibitor Library inhibition screen.


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 of RBC treated with LOPAC compounds palmitoyl-DL-carnitine chloride, calmidazolium chloride, SCH-202676 hydrobromide and rac-2-ethoxy-3-hexadecanamido-1-propylphosphocholinePalmitoyl-DL-carnitine chloride (PKC modulator; A) and calmidazolium chloride (CaM antagonist; B) induced vesiculation assessed by transfer of all events from P2 to P3. SCH-2022676 (GPCR modulator) caused vesiculation (increased number of events in P3) but no shrinkage (no change in P2; C), whereas rac-2-ethoxy-3-hexadecanamido-1-propylphosphocholine had a pronounced effect on RBC preventing the measurement of any events (D). RBCs were treated with the respective compounds for 30 min at 37°C, followed by flow cytometry. All compounds were diluted in DMSO and added at 10 μM final concentration. Plots represent one of three independent measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Scatter plots of RBC treated with LOPAC compounds palmitoyl-DL-carnitine chloride, calmidazolium chloride, SCH-202676 hydrobromide and rac-2-ethoxy-3-hexadecanamido-1-propylphosphocholinePalmitoyl-DL-carnitine chloride (PKC modulator; A) and calmidazolium chloride (CaM antagonist; B) induced vesiculation assessed by transfer of all events from P2 to P3. SCH-2022676 (GPCR modulator) caused vesiculation (increased number of events in P3) but no shrinkage (no change in P2; C), whereas rac-2-ethoxy-3-hexadecanamido-1-propylphosphocholine had a pronounced effect on RBC preventing the measurement of any events (D). RBCs were treated with the respective compounds for 30 min at 37°C, followed by flow cytometry. All compounds were diluted in DMSO and added at 10 μM final concentration. Plots represent one of three independent measurements.
Mentions: After analysis of the induction screens, we identified 12 compounds from LOPAC inducing cell shrinkage/vesiculation and 10 compounds from kinase inhibitor set inducing cell shrinkage/vesiculation. All validated hits from LOPAC and kinase inhibitor induction screens are listed in Tables 1 and 2 respectively. In case of the LOPAC screen, the description includes relevant effects of the compounds not only provided by Sigma (denoted by a star *) but also functions described in literature. Validated hits had a median of the replicates value (Rep median score) higher than 1, meaning they caused an increase in the number of events in P2 compared with controls. Nevertheless, four LOPAC compounds were identified by analysis of RBC scatter alone and not by score value for the following reasons: palmitoyl-DL-carnitine chloride and calmidazolium chloride induced massive vesiculation in RBC leading to a concentration of all events in P3 (Figures 2A and 2B), therefore an increase in P2 could not be used as a parameter. SCH-202676 hydrobromide, on the other hand, induced vesiculation measured by an increase in P3 but no shrinkage (Figure 2C), whereas rac-2-ethoxy-3-hexadecanamido-1-propylphoshocholine's effect on RBC scatter was so strong, no events could be measured (Figure 2D). The adjusted P-value (Padj score) of all hits in the induction screens was ≤0.1, which was considered significant, with the exception of bromoacetyl alprenolol menthane and NNC 55–0396 from the LOPAC (Table 1) and AT7867 and NVP-TAE684 from the Kinase Inhibitor Library (Table 2). Nevertheless, these compounds were included in the validated hit list as they all had Rep median score higher than 1 (Tables 1 and 2). In addition, these compounds induced RBC shrinkage/vesiculation (Figures 4H, 4I, 5J and 5K), measured by a decrease in RBC side scatter, indicating reduced cell size (Figures 4J and 5L). The compounds from the LOPAC library inducing cell shrinkage/vesiculation could be clustered into four functional groups: PKC activity (six compounds including ET-18-OCH3 [34], calcium signalling (two compounds), GPCR signalling (three compounds) and protease activity (one compound) [35] (Figure 3A). Overall these results underscored the potential of our screening method, since the effects of PKC signalling [28], Ca2+ [26] and calpain [27] on RBC have been well studied. In addition, we identified GPCR signalling for the first time to be involved in RBC vesiculation. Figures 2 and 4 illustrate the effects of all LOPAC hits on RBC scatter. Compared with RBC treated with DMSO alone (Figure 4A), it is clear that all validated hits induce RBC shrinkage, measured by a significant increase in the number of events in P2 (Figures 4B–4G), with the exception of bromoacetyl alprenolol menthane and NNC 55–0396 (Figures 4H and 4I), which induced RBC shrinkage measured by a decrease in RBC side scatter (Figure 4J). Since the Kinase Inhibitor Library consisted exclusively of specific kinase inhibitors, we clustered the validated hits according to the family of kinases they target (Figure 3B). Interestingly, we discovered not only compounds targeting kinases with known functions in RBC to induce vesiculation (Table 2), but also compounds inhibiting kinases were described to be expressed in RBC, such as Alk [36] and VEGFR-2 [37]. We could confirm VEGFR-2 was found in RBC with a Western blot (result not shown). Figure 5 shows representative scatter plots of RBC treated with all validated kinase inhibitors. All induction compounds caused a significant increase in P2 compared with treatment with DMSO alone (Figures 5B–5I), with the exception of NVP-TAE684 and AT7867 (Figures 5J and 5K), which induced shrinkage measured by a significant decrease in RBC side scatter (Figure 5L). In addition, NVP-TAE684 treatment lead to vesiculation, measured by an increase in events in P3 (Figure 5J). Moreover, we generated response plots of the hits identified in the kinase inhibitor induction validation screen demonstrating how strong the effect of each compound was (Supplementary Figure). Compound concentrations (100 nM, 1 μM and 1 0μM) were plotted on the x-axis whereas the normalized P2 value was plotted on the y-axis. The response to each compound was represented by a solid line. The closer the line was to the positive control (dotted red line), the stronger the effect was. No compounds inhibiting vesiculation upon ionomycin stimulation were identified during the LOPAC validation inhibition screen and the Kinase Inhibitor Library inhibition screen.

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