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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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RBC shrinkage induced by CX-4945 is mediated via the Gardos channelRBCs were treated with DMSO alone as a control (A), 20 μM TRAM-34 (Gardos channel inhibitor; B), 10 μM CX-4945 (CK2 inhibitor; C) and TRAM-34 followed by CX-4945 (D). RBCs were treated with each compound for 30 min at 37°C followed by flow cytometry. CK2 inhibition lead to RBC shrinkage measured by an increase in events in P2 (C), which could be prevented by blocking the Gardos channel prior to the addition of CX-4945 (D). Plots represent one of six independent measurements. Statistical analysis of CX-4945 effect on RBC shrinkage with or without TRAM-34 treatment (E); results shown represent mean±S.D., n=6; **P<0.01, ***P<0.001, unpaired t test was applied during the analysis.
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Figure 6: RBC shrinkage induced by CX-4945 is mediated via the Gardos channelRBCs were treated with DMSO alone as a control (A), 20 μM TRAM-34 (Gardos channel inhibitor; B), 10 μM CX-4945 (CK2 inhibitor; C) and TRAM-34 followed by CX-4945 (D). RBCs were treated with each compound for 30 min at 37°C followed by flow cytometry. CK2 inhibition lead to RBC shrinkage measured by an increase in events in P2 (C), which could be prevented by blocking the Gardos channel prior to the addition of CX-4945 (D). Plots represent one of six independent measurements. Statistical analysis of CX-4945 effect on RBC shrinkage with or without TRAM-34 treatment (E); results shown represent mean±S.D., n=6; **P<0.01, ***P<0.001, unpaired t test was applied during the analysis.

Mentions: We discovered two compounds inhibiting CK2 (CX-4945 and AS-25245) to induce RBC shrinkage measured by an increase in the number of events in P2 (Figures 5B, 5C, 6C, and 6E). Interestingly, CX-4945 effect on RBC volume was abrogated once the Gardos channel was blocked with the specific inhibitor TRAM-34. We could clearly see that inhibition of CK2 with CX-4945 did not induce RBC shrinkage when the cells were pre-treated with TRAM-34 (Figures 6D and 6E). These results suggest that RBC shrinkage caused by CK2 inhibition is mediated via the Gardos channel. Inhibition of the channel alone did not induce any changes on RBC scatter (Figure 6B).


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)

RBC shrinkage induced by CX-4945 is mediated via the Gardos channelRBCs were treated with DMSO alone as a control (A), 20 μM TRAM-34 (Gardos channel inhibitor; B), 10 μM CX-4945 (CK2 inhibitor; C) and TRAM-34 followed by CX-4945 (D). RBCs were treated with each compound for 30 min at 37°C followed by flow cytometry. CK2 inhibition lead to RBC shrinkage measured by an increase in events in P2 (C), which could be prevented by blocking the Gardos channel prior to the addition of CX-4945 (D). Plots represent one of six independent measurements. Statistical analysis of CX-4945 effect on RBC shrinkage with or without TRAM-34 treatment (E); results shown represent mean±S.D., n=6; **P<0.01, ***P<0.001, unpaired t test was applied during the analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: RBC shrinkage induced by CX-4945 is mediated via the Gardos channelRBCs were treated with DMSO alone as a control (A), 20 μM TRAM-34 (Gardos channel inhibitor; B), 10 μM CX-4945 (CK2 inhibitor; C) and TRAM-34 followed by CX-4945 (D). RBCs were treated with each compound for 30 min at 37°C followed by flow cytometry. CK2 inhibition lead to RBC shrinkage measured by an increase in events in P2 (C), which could be prevented by blocking the Gardos channel prior to the addition of CX-4945 (D). Plots represent one of six independent measurements. Statistical analysis of CX-4945 effect on RBC shrinkage with or without TRAM-34 treatment (E); results shown represent mean±S.D., n=6; **P<0.01, ***P<0.001, unpaired t test was applied during the analysis.
Mentions: We discovered two compounds inhibiting CK2 (CX-4945 and AS-25245) to induce RBC shrinkage measured by an increase in the number of events in P2 (Figures 5B, 5C, 6C, and 6E). Interestingly, CX-4945 effect on RBC volume was abrogated once the Gardos channel was blocked with the specific inhibitor TRAM-34. We could clearly see that inhibition of CK2 with CX-4945 did not induce RBC shrinkage when the cells were pre-treated with TRAM-34 (Figures 6D and 6E). These results suggest that RBC shrinkage caused by CK2 inhibition is mediated via the Gardos channel. Inhibition of the channel alone did not induce any changes on RBC scatter (Figure 6B).

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