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Effects of HC and IFN-α on PV RBC adhesion
expression, as the phosphorylation ratio (P/T), in which P is the measured phosphorylation and T the expression of total Lu/BCAM, was higher during than before HC treat- ment (Figure 4E, bottom panel), indicating that HC both increases the expression of Lu/BCAM and the proportion of phosphorylated molecules.
Discussion
Our recent work showed abnormal expression of sever- al proteins in RBCs of PV patients,19 revealing qualitative alterations in these cells in addition to their increased number in the circulation. The aim of the current study was to address the effects of HC treatment on the expres- sion of erythroid membrane proteins of PV patients using a global proteomic approach.
Our proteomic results obtained with ghosts from 3 pre- post patients showed that HC treatment tends to restore the expression of deregulated membrane proteins. Nevertheless, normal expression of these membrane pro- teins was not reached, suggesting that the duration of the treatment for these patients was not sufficient, which is supported by the results of Calr expression in the group of 11 patients treated with HC for a longer duration.
Since we have shown that Calr overexpression was associated with the presence of JAK2V617F,19 the decreased Calr expression in the HC group could have been the consequence of fewer JAK2V617F-positive clones in the bone marrow of these patients. This was reflected by their lower %V617F when compared to the UT group (median=42.5 and 60% for HC and UT, respec- tively), although the difference was not significant, which is in accordance with a study showing that HC does not appreciably reduce the JAK2V617F allele burden.27 The IFN group also showed lower expression levels of Calr than the UT group, along with a significant decrease of the %V617F (median=15%). This decrease was expected as IFN treatment is known to reduce the JAK2V617F allele burden, with some cases of complete remission.13 Calr is a calcium-binding chaperone that promotes efficient folding of glycoproteins but whose role in circulating RBCs has not been elucidated. Because calcium regulates several erythrocyte functions,28 we believe that the decrease in Calr expression triggered by HC and IFN might be benefi- cial to the calcium homeostasis and subsequent calcium- regulated functions of PV RBCs.
However, despite the tendency to normalize Calr expression, the proteomic analysis showed that HC treat- ment clearly affects the expression of several membrane proteins. This is not the first example of such deregula- tion, as HC is known to induce the neosynthesis of fetal hemoglobin in the erythroid lineage and is used to this purpose in sickle cell disease (SCD) patients.29,30 Among the proteins over-expressed by HC in PV patients, Lu/BCAM and CD147 were also reported to be up-regu- lated in RBCs of sickle cell disease patients treated with HC,31 indicating that the observed effect of HC is not spe- cific to PV. This suggests the triggering of common path- ways by HC independently of the underlying illness, most likely through the activation of gene transcription. This is supported by the findings of Odievre et al.31 showing increased expression of Lu/BCAM in erythroid progeni- tors from SCD patients and healthy donors differentiated in vitro in the presence of HC.31 Moreover, we have previ-
ously shown activation of Lu/BCAM gene transcription by HC in endothelial cells grown in vitro.32
Similar to our previous reports in sSCD, we found that HC significantly increases erythroid Lu/BCAM expression by enhancing both the percentage of Lu/BCAM-positive RBCs and the Lu/BCAM copy number per RBC. The increase in the percentage of Lu/BCAM-positive RBCs indicates that the observed higher expression is not due to the documented increase of the RBC volume consecutive to the HC treatment. Opposite to RBCs from HC-treated SCD patients, those from treated PV patients had increased adhesion to laminin. In SCD, increased Lu/BCAM phosphorylation is achieved in a PKA/cAMP- dependent manner, with patients showing high cAMP lev- els that are decreased during HC treatment.23 In PV, Lu/BCAM phosphorylation is driven by a JAK2V617F- dependent pathway involving Akt, and not PKA, which could explain the difference of HC effects between SCD and PV. HC is a nitric oxide (NO) donor in vivo33-35 and its effect in PV RBCs might be due to Akt activation through an NO-dependent pathway. Indeed, RBCs have been shown to express an active NO synthase (NOS)36 and are thus capable of generating NO and activating downstream effectors like Akt. Such activation of Akt by NO donors has been reported in endothelial cells37 and chick retinal neurons;38 it occurs through the activation of soluble guanylyl cyclase (sGC) and protein kinase G. Interestingly, HC has also been shown to activate sGC in erythroid cells,39 such activation might trigger Akt activity in PV RBCs and lead to Lu/BCAM activation and increased RBC adhesion.
Activation of leukocytes, platelets and endothelial cells is known to promote a prothrombotic state. Markers for such activation have been reported in PV patients, includ- ing activation of polymorphonuclear leukocytes and endothelial cells,40 and increased levels of leukocyte- platelet aggregates.41 A previous report showed that treat- ment of PV patients with HC did not influence these marker levels.42 Our study reveals a new pro-adhesive effect of HC in PV patients that acts by increasing the expression of two adhesion molecules, Lu/BCAM and CD147, and by activating RBC adhesion to laminin. CD147, also named basigin or neurothelin, is a member of the immunoglobulin superfamily that is expressed in var- ious tissues, including brain, leukocytes, endothelial cells, and most tumor cell lines. CD147 is expressed during ery- throid differentiation43 and is carrier molecule for the blood group antigen Oka.44 Its erythroid adhesive function is important during the circulation of RBCs in the spleen.45 It was shown to bind endothelial cells and fibroblasts,46 and to be a receptor essential for erythrocyte invasion by Plasmodium falciparum.47 It is noteworthy that Lu/BCAM and CD147 are adhesion markers linked to progression of solid tumors. Lu/BCAM is over-expressed in carcinomas in vivo and up-regulated following malignant transformation in some cell types.48-51 Likewise, CD147 plays a central role in the progression of many cancers by inducing the secre- tion of matrix metalloproteinases and various cytokines (reviewed by Xiong, Edwards and Zhou52). Several studies have indicated that CD147 is a multifunctional glycopro- tein that inhibits tumor cell anoikis,53 enhances tumor angiogenesis,54 and promotes invasion, metastasis,55 and glycolytic energy metabolism.56 Considering the wide tis- sue distribution of both proteins, and the fact that HC dis- tributes throughout the body reaching approximately all
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