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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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Proposed model of signalling pathways involved in RBC shrinkage and vesiculationInhibition of the depicted kinases induces shrinkage and vesiculation in RBC. CaM antagonism causes ATP depletion and Ca2+ accumulation inside the cell. CK2 inhibition leads to down-modulation of CaM, which in turn activates the Gardos channel leading to K+ efflux and cell shrinkage. Inhibition of GPCR signalling (e.g. P2Y, β-AR) leads to cAMP increase inside the cell and ATP depletion.
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Figure 7: Proposed model of signalling pathways involved in RBC shrinkage and vesiculationInhibition of the depicted kinases induces shrinkage and vesiculation in RBC. CaM antagonism causes ATP depletion and Ca2+ accumulation inside the cell. CK2 inhibition leads to down-modulation of CaM, which in turn activates the Gardos channel leading to K+ efflux and cell shrinkage. Inhibition of GPCR signalling (e.g. P2Y, β-AR) leads to cAMP increase inside the cell and ATP depletion.

Mentions: To our knowledge, a T-type calcium channel, a low voltage channel, has not been described in RBC; however, there are reports of non-selective voltage activated channels in RBC [79] which have been suggested to play a role in increased pathological cation leaks in RBC [80]. Interestingly, our data suggest that T-type calcium channel activity might be related to RBC shrinkage (Figure 4I). Moreover, three compounds from the LOPAC library that we identified as vesiculation inducers are known GPCR antagonists. These are: reactive blue 2 (Basilen blue E-3G, a P2Y receptor antagonist), bromoacetyl alprenolol menthane (a β-AR antagonist; β-blocker) and SCH-202676 hydrobromide, which can act as GPCR agonist as well, since it is described as a general GPCR allosteric modulator. There is evidence in literature of GPCR signalling in RBC [81–83], even though no link to vesiculation has been established yet. P2Y receptors are purinergic GPCR activated by ATP, UDP, ADP, UTP and UDP glucose with various physiological functions including regulation of vascular tone, release of endothelial factors and platelet aggregation [84]. Blood cells express P2Y receptors from different families on their surface, whereas RBC are only known to express P2Y1 [85,86] and P2Y13 [87]. Interestingly, P2Y13 activation by ADP derived from ATP decreases cAMP levels in RBC and prevents ATP release. Furthermore, P2Y13 receptor antagonists stimulate cAMP generation and ATP release from RBC [87]. Our data suggest that treating RBC with a P2Y receptor antagonist can ultimately lead to considerable RBC shrinkage (Figure 4D), possibly due to cAMP signalling and ATP depletion [87]. Moreover, the effects of β-blockers on RBC have been intriguing scientists for a long time. There are reports from the 1970s stating that β-AR antagonists induce RBC K+ release [88] but the mechanisms underlying this phenomenon are still unknown. Several groups have suggested that catecholamines, such as epinephrine, have a positive effect on RBC deformability [89] and filterability [90] via a cAMP-dependent pathway as ATP-depleted RBC were unable to respond to epinephrine [91]. Our results suggest that β-blockers not only reduce RBC deformability [89], but also induce RBC shrinkage (Figure 4H). Furthermore, we discovered several novel kinases to play a role in RBC vesiculation. To our surprise, we identified the Aurora kinase pan inhibitor SNS-314 mesylate [92] as inducer of RBC vesiculation (Figure 5H). Even though Aurora kinases A and B are highly expressed in transferrin receptor (CD71+) early erythroid cells as shown in BioGPS gene annotation portal [93], we did not find any evidence that these kinases are expressed in mature RBC after performing a Western blot (result not shown). Therefore, it is possible that the effect of SNS-314 mesylate observed in RBC is due to an off-target compound effect. For example, SNS-314 has also been shown to inhibit Raf-1 and several RTKs such as high affinity nerve growth factor receptors (TrkA, tropomyosin receptor kinase A and TrkB, tropomyosin receptor kinase B), VEGFR-3, colony stimulating factor 1 receptor, tyrosine-protein kinase receptor UFO (Axl) and Discoidin domain receptor 2 kinase [92]. Interestingly, none of these kinases has been described to have a function in RBC. It is appealing to further investigate what the precise role of these kinases is in RBC signalling and vesiculation. Moreover, the two non-receptor tyrosine kinase (nRTK) inhibitors we discovered to induce RBC vesiculation were NVP-BSK805 (Figure 5D), targeting Jak2 and AP24534 (Ponatinib; Figure 5F), a pan BCR-ABL (breakpoint cluster region protein–Abelson murine leukaemia viral oncogene homologue 1) inhibitor. NVP-BSK805 is a Jak2 kinase inhibitor with effects towards Jak1, Jak3 and Tyk2 kinase as well [94]. Jak2 mediates Epo signalling and is essential during erythropoiesis [95]; however, even though Jak2 is expressed in mature RBC [96], which we could also demonstrate with a western blot (result not shown), its function in RBC is unknown. Interestingly, our results suggest that Jak2 might modulate RBC vesiculation. AP24534 (Ponatinib) is a pan BCR–ABL, including BCR–ABLT315I, inhibitor used in the clinic to treat chronic myeloid leukaemia; however, AP24534 exhibits inhibitory activity towards VEGFR-2, FGFR-1 (fibroblast growth factor receptor-1), scr and Lyn (protein tyrosine kinase Lyn) kinases as well [97]. Reports demonstrate that Lyn is essential for erythropoiesis and Epo-receptor signalling [98]. Furthermore Lyn directly phosphorylates Band-3 [99], thus regulating RBC shape and cytoskeletal rearrangement in healthy RBC. Moreover, Lyn is involved in the pathology of acanthocytosis [100]; nevertheless its role in RBC vesiculation has not been addressed. Another target of AP24534 is the nRTK VEGFR-2. Expression of VEGFR-2 in RBC is debatable. Sachanonta et al. [37] have shown that malaria-infected RBC and RBC stain positive for VEGFR-2, even though the authors speculated that the detected expression might be due to passive uptake of the receptor by RBC from serum. We have confirmed that VEGFR-2 is present in RBC by a Western blot (result not shown) and our data suggest that VEGFR-2 inhibition regulates RBC volume. VEGFR-2 activates PI3K–Akt pathway and PLC (phospholipase C)–PKC pathway [101], thus it is tempting to speculate that VEGFR-2 is involved in vesiculation as well since downstream targets of VEGFR-2 are known to have various roles in RBC signalling. The only RTK inhibitor we found to induce RBC vesiculation was NVP-TAE684, an Alk RTK inhibitor [102]. Alk kinase is upstream of various signalling pathways such as the MAPK–ERK, the Jak–STAT and the PI3K–Akt pathway [103]. Alk kinase has been described to be expressed in RBC only recently [36] and since our screen results showed that Alk inhibition induced RBC vesiculation (Figure 5J), it is possible that this kinase is involved in RBC signalling as well. In addition, we discovered two inhibitors of CK2: CX-4945 (Silmitasertib), a specific inhibitor [104] and AS-252424, inhibitor at 10 μM [77] that induced RBC shrinkage (Figures 5B an 5C). CK2 is highly expressed in RBC and has been shown to play a role in immune adherence clearance [105] and to mediate membrane deformability upon complement receptor 1 ligation [106]. Interestingly, in neurons CK2 binds to the Ca2+-activated potassium channel SK2 (small conductance calcium-activated potassium channel 2), thus directly regulating its function [107]. Since SK2 requires CaM signalling for proper functioning, CK2 phosphorylation of CaM abrogates SK2 channel's sensitivity to calcium leading to SK2 inactivation [107]. Interestingly, the Gardos channel, as a small conductance calcium-activated potassium channel, shares homology with the other SK channels [41]. Involvement of CK2 in the activity of the Gardos channel in RBC has not been demonstrated. We suggest that RBC shrinkage induced by CK2 inhibition is mediated via the Gardos channel since blocking of the channel with the specific inhibitor TRAM-34 prevents shrinkage induced by CX-4945 (Figure 6). We speculate that, similar to what is observed in neurons, CK2 might be co-assembling with the Gardos channel, modulating its function [107]. Inhibition of CK2 would prevent CaM phosphorylation, which could lead to constitutive activation of the potassium channel due to increased Ca2+ sensitivity [108], resulting in cell shrinkage. Certainly, a demonstration of the direct interaction between CK2 and the Gardos channel is necessary to further confirm their concerted role in RBC vesiculation. Lastly, we did not identify any compounds inhibiting vesiculation upon ionomycin stimulation. One reason for the lack of hits in the inhibition screens could be the challenging task to abrogate the strong effect of RBC vesiculation induced by ionomycin. In conclusion, we discovered several novel signalling cascades to be involved in RBC vesiculation, including GPCR signalling, the PI3K–Akt pathway, the Raf–MEK–ERK pathway, and the Jak–STAT pathway. Moreover, we suggest for the first time a role of CK2, Alk kinase and VEGFR-2 in RBC shrinkage and vesiculation (Figure 7). We cannot exclude the possibility of redundancy in some of these pathways and more research is needed to elucidate the exact functions of these cascades in RBC vesiculation and the storage lesion.


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)

Proposed model of signalling pathways involved in RBC shrinkage and vesiculationInhibition of the depicted kinases induces shrinkage and vesiculation in RBC. CaM antagonism causes ATP depletion and Ca2+ accumulation inside the cell. CK2 inhibition leads to down-modulation of CaM, which in turn activates the Gardos channel leading to K+ efflux and cell shrinkage. Inhibition of GPCR signalling (e.g. P2Y, β-AR) leads to cAMP increase inside the cell and ATP depletion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Proposed model of signalling pathways involved in RBC shrinkage and vesiculationInhibition of the depicted kinases induces shrinkage and vesiculation in RBC. CaM antagonism causes ATP depletion and Ca2+ accumulation inside the cell. CK2 inhibition leads to down-modulation of CaM, which in turn activates the Gardos channel leading to K+ efflux and cell shrinkage. Inhibition of GPCR signalling (e.g. P2Y, β-AR) leads to cAMP increase inside the cell and ATP depletion.
Mentions: To our knowledge, a T-type calcium channel, a low voltage channel, has not been described in RBC; however, there are reports of non-selective voltage activated channels in RBC [79] which have been suggested to play a role in increased pathological cation leaks in RBC [80]. Interestingly, our data suggest that T-type calcium channel activity might be related to RBC shrinkage (Figure 4I). Moreover, three compounds from the LOPAC library that we identified as vesiculation inducers are known GPCR antagonists. These are: reactive blue 2 (Basilen blue E-3G, a P2Y receptor antagonist), bromoacetyl alprenolol menthane (a β-AR antagonist; β-blocker) and SCH-202676 hydrobromide, which can act as GPCR agonist as well, since it is described as a general GPCR allosteric modulator. There is evidence in literature of GPCR signalling in RBC [81–83], even though no link to vesiculation has been established yet. P2Y receptors are purinergic GPCR activated by ATP, UDP, ADP, UTP and UDP glucose with various physiological functions including regulation of vascular tone, release of endothelial factors and platelet aggregation [84]. Blood cells express P2Y receptors from different families on their surface, whereas RBC are only known to express P2Y1 [85,86] and P2Y13 [87]. Interestingly, P2Y13 activation by ADP derived from ATP decreases cAMP levels in RBC and prevents ATP release. Furthermore, P2Y13 receptor antagonists stimulate cAMP generation and ATP release from RBC [87]. Our data suggest that treating RBC with a P2Y receptor antagonist can ultimately lead to considerable RBC shrinkage (Figure 4D), possibly due to cAMP signalling and ATP depletion [87]. Moreover, the effects of β-blockers on RBC have been intriguing scientists for a long time. There are reports from the 1970s stating that β-AR antagonists induce RBC K+ release [88] but the mechanisms underlying this phenomenon are still unknown. Several groups have suggested that catecholamines, such as epinephrine, have a positive effect on RBC deformability [89] and filterability [90] via a cAMP-dependent pathway as ATP-depleted RBC were unable to respond to epinephrine [91]. Our results suggest that β-blockers not only reduce RBC deformability [89], but also induce RBC shrinkage (Figure 4H). Furthermore, we discovered several novel kinases to play a role in RBC vesiculation. To our surprise, we identified the Aurora kinase pan inhibitor SNS-314 mesylate [92] as inducer of RBC vesiculation (Figure 5H). Even though Aurora kinases A and B are highly expressed in transferrin receptor (CD71+) early erythroid cells as shown in BioGPS gene annotation portal [93], we did not find any evidence that these kinases are expressed in mature RBC after performing a Western blot (result not shown). Therefore, it is possible that the effect of SNS-314 mesylate observed in RBC is due to an off-target compound effect. For example, SNS-314 has also been shown to inhibit Raf-1 and several RTKs such as high affinity nerve growth factor receptors (TrkA, tropomyosin receptor kinase A and TrkB, tropomyosin receptor kinase B), VEGFR-3, colony stimulating factor 1 receptor, tyrosine-protein kinase receptor UFO (Axl) and Discoidin domain receptor 2 kinase [92]. Interestingly, none of these kinases has been described to have a function in RBC. It is appealing to further investigate what the precise role of these kinases is in RBC signalling and vesiculation. Moreover, the two non-receptor tyrosine kinase (nRTK) inhibitors we discovered to induce RBC vesiculation were NVP-BSK805 (Figure 5D), targeting Jak2 and AP24534 (Ponatinib; Figure 5F), a pan BCR-ABL (breakpoint cluster region protein–Abelson murine leukaemia viral oncogene homologue 1) inhibitor. NVP-BSK805 is a Jak2 kinase inhibitor with effects towards Jak1, Jak3 and Tyk2 kinase as well [94]. Jak2 mediates Epo signalling and is essential during erythropoiesis [95]; however, even though Jak2 is expressed in mature RBC [96], which we could also demonstrate with a western blot (result not shown), its function in RBC is unknown. Interestingly, our results suggest that Jak2 might modulate RBC vesiculation. AP24534 (Ponatinib) is a pan BCR–ABL, including BCR–ABLT315I, inhibitor used in the clinic to treat chronic myeloid leukaemia; however, AP24534 exhibits inhibitory activity towards VEGFR-2, FGFR-1 (fibroblast growth factor receptor-1), scr and Lyn (protein tyrosine kinase Lyn) kinases as well [97]. Reports demonstrate that Lyn is essential for erythropoiesis and Epo-receptor signalling [98]. Furthermore Lyn directly phosphorylates Band-3 [99], thus regulating RBC shape and cytoskeletal rearrangement in healthy RBC. Moreover, Lyn is involved in the pathology of acanthocytosis [100]; nevertheless its role in RBC vesiculation has not been addressed. Another target of AP24534 is the nRTK VEGFR-2. Expression of VEGFR-2 in RBC is debatable. Sachanonta et al. [37] have shown that malaria-infected RBC and RBC stain positive for VEGFR-2, even though the authors speculated that the detected expression might be due to passive uptake of the receptor by RBC from serum. We have confirmed that VEGFR-2 is present in RBC by a Western blot (result not shown) and our data suggest that VEGFR-2 inhibition regulates RBC volume. VEGFR-2 activates PI3K–Akt pathway and PLC (phospholipase C)–PKC pathway [101], thus it is tempting to speculate that VEGFR-2 is involved in vesiculation as well since downstream targets of VEGFR-2 are known to have various roles in RBC signalling. The only RTK inhibitor we found to induce RBC vesiculation was NVP-TAE684, an Alk RTK inhibitor [102]. Alk kinase is upstream of various signalling pathways such as the MAPK–ERK, the Jak–STAT and the PI3K–Akt pathway [103]. Alk kinase has been described to be expressed in RBC only recently [36] and since our screen results showed that Alk inhibition induced RBC vesiculation (Figure 5J), it is possible that this kinase is involved in RBC signalling as well. In addition, we discovered two inhibitors of CK2: CX-4945 (Silmitasertib), a specific inhibitor [104] and AS-252424, inhibitor at 10 μM [77] that induced RBC shrinkage (Figures 5B an 5C). CK2 is highly expressed in RBC and has been shown to play a role in immune adherence clearance [105] and to mediate membrane deformability upon complement receptor 1 ligation [106]. Interestingly, in neurons CK2 binds to the Ca2+-activated potassium channel SK2 (small conductance calcium-activated potassium channel 2), thus directly regulating its function [107]. Since SK2 requires CaM signalling for proper functioning, CK2 phosphorylation of CaM abrogates SK2 channel's sensitivity to calcium leading to SK2 inactivation [107]. Interestingly, the Gardos channel, as a small conductance calcium-activated potassium channel, shares homology with the other SK channels [41]. Involvement of CK2 in the activity of the Gardos channel in RBC has not been demonstrated. We suggest that RBC shrinkage induced by CK2 inhibition is mediated via the Gardos channel since blocking of the channel with the specific inhibitor TRAM-34 prevents shrinkage induced by CX-4945 (Figure 6). We speculate that, similar to what is observed in neurons, CK2 might be co-assembling with the Gardos channel, modulating its function [107]. Inhibition of CK2 would prevent CaM phosphorylation, which could lead to constitutive activation of the potassium channel due to increased Ca2+ sensitivity [108], resulting in cell shrinkage. Certainly, a demonstration of the direct interaction between CK2 and the Gardos channel is necessary to further confirm their concerted role in RBC vesiculation. Lastly, we did not identify any compounds inhibiting vesiculation upon ionomycin stimulation. One reason for the lack of hits in the inhibition screens could be the challenging task to abrogate the strong effect of RBC vesiculation induced by ionomycin. In conclusion, we discovered several novel signalling cascades to be involved in RBC vesiculation, including GPCR signalling, the PI3K–Akt pathway, the Raf–MEK–ERK pathway, and the Jak–STAT pathway. Moreover, we suggest for the first time a role of CK2, Alk kinase and VEGFR-2 in RBC shrinkage and vesiculation (Figure 7). We cannot exclude the possibility of redundancy in some of these pathways and more research is needed to elucidate the exact functions of these cascades in RBC vesiculation and the storage lesion.

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