Decreased sickle red blood cell adhesion to laminin by hydroxyurea is associated with inhibition of Lu/BCAM protein phosphorylation

Bartolucci, Pablo; Chaar, Vicky; Picot, Julien; Bachir, Dora; Habibi, Anoosha; Fauroux, Christine; Galactéros, Frédéric; Colin, Yves; Kim, Caroline Le Van; Nemer, Wassim El

doi:10.1182/blood-2009-12-257444

Cited by 69 publications

(76 citation statements)

References 46 publications

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“…The extra 40 a.a. of Lu comprise phosphorylated serines, 12 which have been shown to play a critical role in Lu/BCAM-mediated RBC adhesion to laminin in sickle cell disease. 13,14 We showed in our previous work that PV RBC adhesion to laminin was associated with Lu/BCAM hyperphosphorylation. Lu/BCAM phosphorylation also was strongly increased in a K562 cell line coexpressing Lu and JAK2V617F, suggesting that erythroid activation of Lu/BCAM could result from the presence of JAK2V617F in PV RBCs.…”

Section: Introductionmentioning

confidence: 93%

“…12,13 Inhibitors were used under the conditions described for the adhesion assays. In vitro phosphorylation of Lu cytoplasmic domain by purified Akt (Sigma-Aldrich) was performed as described 12 by the use of 5 g of glutathione-S-transferase (GST) fusion proteins in which a serine of the linker region, potentially targeted by Akt, was replaced by alanine.…”

Section: Phosphorylation Assaysmentioning

confidence: 99%

“…12,13 Akt activity quantification Akt activity was measured in cell lysates of HEL cells or RBCs using the Akt kinase activity kit (Enzo Life Sciences). Protein quantification after cell lysis was performed with the BCA Protein Assay Kit (Pierce-Thermo Scientific).…”

Section: Flow Cytometrymentioning

confidence: 99%

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JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway

Grandis

Cambot

Wautier

et al. 2013

Blood

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Key Points• In polycythemia vera RBCs, JAK2V617F is able to activate an adhesion molecule through a Rap1/Akt pathway, despite the absence of EpoR.• In polycythemia vera, the abnormal adhesion of RBCs to laminin is due to the phosphorylation of Lu/BCAM by a JAK2V617F/Rap1/Akt pathway.Polycythemia vera (PV) is characterized by an increased RBC mass, spontaneous erythroid colony formation, and the JAK2V617F mutation. PV is associated with a high risk of mesenteric and cerebral thrombosis. PV RBC adhesion to endothelial laminin is increased and mediated by phosphorylated erythroid Lu/BCAM. In the present work, we investigated the mechanism responsible for Lu/BCAM phosphorylation in the presence of JAK2V617F using HEL and BaF3 cell lines as well as RBCs from patients with PV. High levels of Rap1-GTP were found in HEL and BaF3 cells expressing JAK2V617F compared with BaF3 cells with wild-type JAK2. This finding was associated with increased Akt activity, Lu/BCAM phosphorylation, and cell adhesion to laminin that were inhibited by the dominant-negative Rap1S17N or by the specific Rap1 inhibitor GGTI-298. Surprisingly, knocking-down EpoR in HEL cells did not alter Akt activity or cell adhesion to laminin. Our findings reveal a novel EpoR-independent IntroductionPolycythemia vera (PV) is the most common myeloproliferative neoplasm. 1 It is characterized by erythropoietin-independent erythroid colony formation in vitro, which is associated with somatic gain-of-function mutations in the gene encoding the tyrosine kinase JAK2. [2][3][4][5] The most frequent mutation is a substitution from valine to phenylalanine at position 617, which renders JAK2 constitutively active, leading to uncontrolled cell proliferation in the erythroid lineage and resulting in increased red cell mass. Patients with PV exhibit increased platelet and leukocyte counts and have a high risk of thrombosis, which is considered a major cause of mortality and morbidity in this disease. 6 In a previous work, we showed that PV RBCs were abnormally adherent to endothelial cells because of the interaction between erythroid Lutheran/basal cell-adhesion molecule (Lu/BCAM) and endothelial laminin. 7 Lu/BCAM is an adhesion protein of the immunoglobulin superfamily with a large tissue distribution. 8,9 It is the unique erythroid receptor of laminin ␣5 chain, constituent of laminin 511/521 isoforms, and a major component of the extracellular matrix. 10,11 Lu/BCAM is expressed as 2 isoforms of 85 and 78 kDa, named Lu and Lu(v13), respectively. 9,10 The 2 isoforms differ only by the length of their cytoplasmic domain, which is composed of 19 a.a. for Lu(v13) and 59 a.a. for Lu. The extra 40 a.a. of Lu comprise phosphorylated serines, 12 which have been shown to play a critical role in Lu/BCAM-mediated RBC adhesion to laminin in sickle cell disease. 13,14 We showed in our previous work that PV RBC adhesion to laminin was associated with Lu/BCAM hyperphosphorylation. Lu/BCAM phosphorylation also was strongly increased in a K562 cell line coexpressing Lu and JAK2V617F, ...

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Section: Introductionmentioning

confidence: 93%

Section: Phosphorylation Assaysmentioning

confidence: 99%

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JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway

Grandis

Cambot

Wautier

et al. 2013

Blood

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“…There have been other therapeutic agents that have been shown to increase production of HbF as well; however, none of them is currently approved by the FDA (8,9). Other effects of HU administration in SCD patients on RBCs include larger cell volumes (10), increased antioxidant activity (11), reduced adhesiveness (12)(13)(14), and greater filterability (15). Mechanisms underlying these and other beneficial effects of the drug affecting leukocytes and the endothelium (16)(17)(18) have not been fully elucidated.…”

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confidence: 99%

Cellular normoxic biophysical markers of hydroxyurea treatment in sickle cell disease

Hosseini

Abidi

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Hydroxyurea (HU) has been used clinically to reduce the frequency of painful crisis and the need for blood transfusion in sickle cell disease (SCD) patients. However, the mechanisms underlying such beneficial effects of HU treatment are still not fully understood. Studies have indicated a weak correlation between clinical outcome and molecular markers, and the scientific quest to develop companion biophysical markers have mostly targeted studies of blood properties under hypoxia. Using a common-path interferometric technique, we measure biomechanical and morphological properties of individual red blood cells in SCD patients as a function of cell density, and investigate the correlation of these biophysical properties with drug intake as well as other clinically measured parameters. Our results show that patient-specific HU effects on the cellular biophysical properties are detectable at normoxia, and that these properties are strongly correlated with the clinically measured mean cellular volume rather than fetal hemoglobin level.

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“…One has also to keep in mind the importance of posttranslational phosphorylation-mediated regulation of the adhesion properties of some adhesion molecules. In this context, of particular interest is the recent observation by Bartolucci et al 52 that the decreased RBC adhesion to laminin in SCD patients treated with HC is associated with inhibition of erythroid Lu/BCAM protein phosphorylation. Thus, it is clear that in the future, analysis of HC action on adhesion processes should no longer be restricted to mRNA/protein levels.…”

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confidence: 99%

Differential modulation of adhesion molecule expression by hydroxycarbamide in human endothelial cells from the micro- and macrocirculation: potential implications in sickle cell disease vasoocclusive events

Laurance¹,

Lansiaux²,

Pellay³

et al. 2011

Haematologica

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BackgroundAll the cellular partners of the vascular system and especially endothelial cells are involved in the pathophysiology of the vasoocclusive crises associated with sickle cell disease. In sickle cell disease, circulating cells adhere abnormally to endothelial cells in a chronic pro-inflammatory context. Hydroxycarbamide is the only drug with demonstrated efficacy to reduce the frequency of vasoocclusive crises. Here, we investigated the effects of hydroxycarbamide and/or cytokines on the expression of genes related to adhesion events in endothelial cells from three different vascular sites. Design and MethodsEndothelial cells representative of the macro-(HUVEC) or microcirculation (TrHBMEC and HPMEC) were grown in the presence or absence of hydroxycarbamide and/or cytokines (TNFα and IFNγ). Expression of genes encoding adhesion proteins was analyzed by RQ-PCR, ELISA, flow cytometry, in situ ELISA for extracellular matrix proteins, and Western blot. ResultsIn cells from the microcirculation, expression of TSP-1, vWF, and PECAM-1 genes was decreased by hydroxycarbamide and/or cytokine treatment at the mRNA level. In the macrocirculation their expression was unaffected or increased. Hydroxycarbamide significantly decreased vWF incorporated in the TrHBMEC extracellular matrix. CD36 mRNA was strongly down-regulated by cytokines in HPMEC, the only cell type in which it is expressed. Hydroxycarbamide decreased soluble PECAM-1 in HUVEC supernatants. ConclusionsOur results highlight the heterogeneity of vascular endothelial cell responses to hydroxycarbamide and/or cytokines depending upon their origin. They also suggest that hydroxycarbamide has an anti-adhesogenic effect on endothelial cells, but by mechanisms which could vary according to their macro-or microcirculation and organ origin.Key words: sickle cell disease, adhesion, hydroxycarbamide, vascular endothelial cells. disease vasoocclusive events. Haematologica 2011;96(4):534-542. doi:10.3324/haematol.2010 This is an open-access paper. Citation: Laurance S, Lansiaux P, Pellay F-X, Hauchecorne M, Benecke A, Elion J, and Lapoumeroulie C. Differential modulation of adhesion molecule expression by hydroxycarbamide in human endothelial cells from the micro-and macrocirculation: potential implications in sickle cell

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Decreased sickle red blood cell adhesion to laminin by hydroxyurea is associated with inhibition of Lu/BCAM protein phosphorylation

Cited by 69 publications

References 46 publications

JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway

JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway

Cellular normoxic biophysical markers of hydroxyurea treatment in sickle cell disease

Differential modulation of adhesion molecule expression by hydroxycarbamide in human endothelial cells from the micro- and macrocirculation: potential implications in sickle cell disease vasoocclusive events

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