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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 Rap1/Akt signaling pathway that is activated by JAK2V617F in circulating PV RBCs and responsible for Lu/BCAM activation. This new characteristic of JAK2V617F could play a critical role in initiating abnormal interactions among circulating and endothelial cells in patients with PV.

International audience; Maturation of newly formed vessels is a multistep phenomenon during which functional endothelial barriers are established. Disruption of vessel integrity is an important feature in many physiological and pathological processes. We previously reported that lymphoblastic leukemia-derived sequence 1 (LYL1) is required for the late stages of postnatal angiogenesis to limit the formation of new blood vessels, notably by regulating the activity of the small GTPase Rap1. In this study, we show that LYL1 is also required during the formation of the mature endothelial barrier in the lungs of adult mice. Specifically, LYL1 knockdown in human endothelial cells downregulated the expression of ARHGAP21 and ARHGAP24, which encode two Rho GTPase-activating proteins, and this was correlated with increased RhoA activity and reorganization of the actin cytoskeleton into stress fibers. Importantly, in lungs of Lyl1-deficient mice, both vascular endothelial (VE)-cadherin and p120-catenin were poorly recruited to endothelial adherens junctions, indicative of defective cell-cell junctions. Consistent with this, higher Evans blue dye extravasation, edema, and leukocyte infiltration in the lung parenchyma of Lyl1-/- mice than in wild-type littermates confirmed that lung vascular permeability is constitutively elevated in Lyl1-/- adult mice. Our data show that LYL1 acts as a stabilizing signal for adherens junction formation by operating upstream of VE-cadherin and of the two GTPases Rap1 and RhoA. As increased vascular permeability is a key feature and a major mechanism of acute respiratory distress syndrome, molecules that regulate LYL1 activity could represent additional tools to modify the endothelial barrier permeability.

International audience; The cAMP-binding protein Epac is a therapeutic target for the treatment of various diseases such as cardiac hypertrophy and tumor invasion. This points out the importance to develop Epac inhibitors to better understand the involvement of these cAMP sensors in physiology and pathophysiology. Here, we have developed a functional fluorescence-based high-throughput assay with a Z' value around 0.7 for screening Epac-specific antagonists. We identified an Epac1 inhibitor compound named CE3F4 that blocked Epac1 guanine nucleotide exchange activity toward its effector Rap1 both in cell-free systems and in intact cells. CE3F4 is a tetrahydroquinoline analog that fails to influence protein kinase A holoenzyme activity. CE3F4 inhibited neither the interaction of Rap1 with Epac1 nor directly the GDP exchange on Rap1. The kinetics of inhibition by CE3F4 indicated that this compound did not compete for binding of agonists to Epac1 and suggested an uncompetitive inhibition mechanism with respect to Epac1 agonists. A structure-activity study showed that the formyl group on position 1 and the bromine atom on position 5 of the tetrahydroquinoline skeleton were important for CE3F4 to exert its inhibitory activity. Finally, CE3F4 inhibited Rap1 activation in living cultured cells, following Epac activation by either 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac-selective agonist, or isoprenaline, a non-selective β-adrenergic receptor agonist. Our study shows that CE3F4 and related compounds may serve as a basis for the development of new therapeutic drugs.

The MAPK ERK1/2 cascade regulates all the critical cellular functions, and in many pathological situations, these regulatory processes are perturbed. It has been clearly established that this cascade is an integrative point in the control of the pituitary functions exerted by various extracellular signals. In particular, ERK1/2 cross talk with the cAMP pathway is determinant in the control of somatolactotroph hormonal secretion exerted via neuropeptide receptors. GH-secreting adenomas are characterized by frequent cAMP pathway alterations, such as constitutive activation of the α-subunit of the heterotrimeric Gs protein (the gsp oncogene), overexpression of Gsα, and changes in the protein kinase A regulatory subunits. However, it has not yet been established exactly how these alterations result in GH-secreting adenomas or how the ERK1/2 cascade contributes to the process of GH-secreting adenoma tumorigenesis. In this study on the conditional gsp-oncogene-expressing GH4C1 cell line, expression of the gsp oncogene, which was observed in up to 40% of GH-secreting adenomas, was found to induce sustained ERK1/2 activation, which required activation of the protein kinase A and the GTPases Ras and Rap1. All these signaling components contribute to the chronic activation of the human prolactin promoter. The data obtained here show that Ras plays a crucial role in these processes: in a physiopathological context, i.e. in the presence of the gsp oncogene, it switched from being a repressor of the cAMP/ protein kinase A ERK-sensitive prolactin gene control exerted by neuropeptides to an activator of the prolactin promoter.

International audience; Cerebral cavernous malformations (CCM) are common vascular malformations with an unpredictable risk of hemorrhage, the consequences of which range from headache to stroke or death. Three genes, CCM1, CCM2 and CCM3, have been linked to the disease. The encoded CCM proteins interact with each other within a large protein complex. Within the past 2 years, a plethora of new data has emerged on the signaling pathways in which CCM proteins are involved. CCM proteins regulate diverse aspects of endothelial cell morphogenesis and blood vessel stability such as cell-cell junctions, cell shape and polarity, or cell adhesion to the extracellular matrix. Although fascinating, a global picture is hard to depict because little is known about how these pathways coordinate to orchestrate angiogenesis. Here we present what is known about the structural domain organization of CCM proteins, their association as a ternary complex and their subcellular localization. Numerous CCM partners have been identified using two-hybrid screens, genetic analyses or proteomic studies. We focus on the best-characterized partners and review data on the signaling pathways they regulate as a step towards a better understanding of the etiology of CCM disease.