C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation

[Background]: Megakaryopoiesis allows platelet formation, which is necessary for coagulation, also playing an important role in different pathologies. However, this process remains to be fully characterized. C3G, an activator of Rap1 GTPases, is involved in platelet activation and regulates several differentiation processes. [Methods]: We evaluated C3G function in megakaryopoiesis using transgenic mouse models where C3G and C3GΔCat (mutant lacking the GEF domain) transgenes are expressed exclusively in megakaryocytes and platelets. In addition, we used different clones of K562, HEL and DAMI cell lines with overexpression or silencing of C3G or GATA-1. [Results]: We found that C3G participates in the differentiation of immature hematopoietic cells to megakaryocytes. Accordingly, bone marrow cells from transgenic C3G, but not those from transgenic C3GΔCat mice, showed increased expression of the differentiation markers CD41 and CD61, upon thrombopoietin treatment. Furthermore, C3G overexpression increased the number of CD41+ megakaryocytes with high DNA content. These results are supported by data obtained in the different models of megakaryocytic cell lines. In addition, it was uncovered GATA-1 as a positive regulator of C3G expression. Moreover, C3G transgenic megakaryocytes from fresh bone marrow explants showed increased migration from the osteoblastic to the vascular niche and an enhanced ability to form proplatelets. Although the transgenic expression of C3G in platelets did not alter basal platelet counts, it did increase slightly those induced by TPO injection in vivo. Moreover, platelet C3G induced adipogenesis in the bone marrow under pathological conditions. [Conclusions]: All these data indicate that C3G plays a significant role in different steps of megakaryopoiesis, acting through a mechanism dependent on its GEF activity.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness [SAF2013–48210-C2–1-R and SAF2016–76588-C2–2-R to CG, SAF2013–48210-C2–2-R and SAF2016–76588-C2–1-R to AP], and by two grants from the Council of Education of Junta de Castilla y León, Spain [SA157A12–1 and SA017U16 to CG]. All funding was cosponsored by the European FEDER Program

C3G promotes a selective release of angiogenic factors from activated mouse platelets to regulate angiogenesis and tumor metastasis

[EN]Previous observations indicated that C3G (RAPGEF1) promotes α-granule release, evidenced by the increase in P-selectin exposure on the platelet surface following its activation. The goal of the present study is to further characterize the potential function of C3G as a modulator of the platelet releasate and its implication in the regulation of angiogenesis. Proteomic analysis revealed a decreased secretion of anti-angiogenic factors from activated transgenic C3G and C3GΔCat platelets. Accordingly, the secretome from both transgenic platelets had an overall pro-angiogenic effect as evidenced by an in vitro capillary-tube formation assay with HUVECs (human umbilical vein endothelial cells) and by two in vivo models of heterotopic tumor growth. In addition, transgenic C3G expression in platelets greatly increased mouse melanoma cells metastasis. Moreover, immunofluorescence microscopy showed that the pro-angiogenic factors VEGF and bFGF were partially retained into α-granules in thrombin- and ADP-activated mouse platelets from both, C3G and C3GΔCat transgenic mice. The observed interaction between C3G and Vesicle-associated membrane protein (Vamp)-7 could explain these results. Concomitantly, increased platelet spreading in both transgenic platelets upon thrombin activation supports this novel function of C3G in α-granule exocytosis. Collectively, our data point out to the co-existence of Rap1GEF-dependent and independent mechanisms mediating C3G effects on platelet secretion, which regulates pathological angiogenesis in tumors and other contexts. The results herein support an important role for platelet C3G in angiogenesis and metastasis

Cuerpo y simbolismo amerindio: diseño museográfico y docente de la colección complutense del Museo de Arqueología y Etnología de América (MAEA, Facultad de Geografía e Historia) referida al cuerpo humano

Depto. de Historia de América y Medieval y Ciencias HistoriográficasFac. de Geografía e HistoriaFALSEsubmitte

Papel de C3G plaquetario en angiogénesis y metástasis tumoral

[ES]Observaciones previas indican que la proteína C3G (RapGEF1) promueve la activación plaquetaria y la liberación de los gránulos-α, según se ha evidenciado por el incremento de la expresión de P-selectina en la superficie de la plaqueta, tras la activación. El objetivo del presente estudio fue estudiar la función potencial de C3G como modulador del secretoma plaquetario y su implicación en la regulación de la angiogénesis. El análisis proteómico de los secretomas reveló una disminución de la secreción de factores anti-angiogénicos en las plaquetas transgénicas para C3G y en las plaquetas transgénicas para una versión mutante de C3G que no posee actividad catalítica GEF (C3GΔCat). En concordancia, el secretoma de ambas plaquetas transgénicas presentó un efecto neto pro-angiogénico, según se evidenció en un ensayo de formación de capilares in vitro utilizando células HUVEC (células endoteliales de la vena umbilical humana), y en dos modelos in vivo de crecimiento de tumores heterotópicos en ratón. Adicionalmente, la expresión transgénica de C3G en plaquetas incrementó considerablemente el número de metástasis de células de melanoma de ratón. Por otra parte, ensayos de microscopía de inmunofluorescencia mostraron que los factores pro-angiogénicos VEGF y bFGF se encontraban parcialmente retenidos en los gránulos-α de las plaquetas de ratones de ambos genotipos transgénicos, tras la activación con ADP o trombina. La observación de la interacción entre C3G y una de las proteínas asociadas a la membrana de los gránulos (VAMP7) podría explicar estos resultados. En concordancia, la modulación positiva de la extensión sobre sustrato en ambas plaquetas transgénicas, tras la activación con trombina, apoya esta nueva función de C3G en la exocitosis de los gránulos-α. En conjunto, nuestros datos sugieren la co-existencia de mecanismos independientes y dependientes de la actividad Rap1GEF de C3G mediante los cuales esta proteína modula la secreción plaquetaria, regulando la angiogénesis patológica en tumores y en otros contextos fisiológicos. Los datos presentados apoyan un papel importante de C3G plaquetario en angiogénesis y metástasis

C3G regulates megakaryocytic differentiation

Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015.C3G (Crk-SH3-binding Guanine nucleotide Exchange Factor) is an activator of Ras family member Rap1/2. Several studies suggest a role for C3G in cell differentiation; C3G is induced during neural differentiation, as well as during differentiation of monocytic cells to macrophages. Similarly, enhancement in C3G protein and its association with Crk is required for adipocyte differentiation. Some evidences suggest a participation of C3G in the differentiation of megakaryocytes to platelets through Rap1 activation, although its potential role in early stages of megakaryocytic differentiation has not been addressed yet. Using erythroleukemic cell lines K562 and HEL we have found that C3G plays a role in the differentiation of immature cells to megakaryocytes, as overexpression of C3G increases the expression of megakaryocytic markers CD41 and CD61, while decreases erythroid marker glycophorin A. In contrast, the expression of CD41/CD61 decreases in C3G silenced cells. Morphology and polyploidisation analysis support the hypothesis of a positive role of C3G in megakaryocytic differentiation. This effect of C3G in megakaryopoiesis is dependent on PKC pathway as bisyndolylmaleimide completely abrogated it. Additionally, studies in double C3G/p38aMAPK K562 knockdown clones suggest a relationship between these two proteins in the regulation of K562 megakaryocytic differentiation. In concordance with the studies in cell lines, TPOstimulated bone marrow cells from megakaryocyte- and platelet-specific C3G transgenic mice showed higher levels of CD41/CD61 as compared to wild type cells. In contrast, bone marrow cells from C3GDCat transgenic mice showed lower levels of these megakaryocytic markers, indicating that the effect of C3G in megakaryocytic differentiation is Rap1-dependent.Peer reviewe

C3G regulates megakaryocytic differentiation and pro-platelet formation in mice

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.C3G is an activator of members of the Rap subfamily of Ras proteins. Several studies suggest that C3G plays a role in different processes of differentiation acting through its main target Rap1. C3G is induced during neural differentiation and monocytic differentiation to macrophages. Similarly, an increase in C3G protein levels and its association with Crk is required for adipocyte differentiation. There are also evidences supporting the involvement of C3G in the differentiation of megakaryocytes to platelets through Rap1 activation, although its function in early stages of megakaryocytic differentiation has not yet been addressed. Using transgenic mouse models for C3G and C3GΔCat (C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 gene promoter, we have found that C3G plays a role in the differentiation of immature hematopoietic cells to megakaryocytes. Bone marrow cells from transgenic C3G, but not those from transgenic C3GΔCat mice, showed increased CD41 and CD61 expression upon thrombopoietin (TPO) treatment. Polyploidization analysis supports the hypothesis of a positive role of C3G in megakaryocytic differentiation. Thus, C3G overexpression increased the levels of CD41+ megakaryocytes with 16N and 32N ploidy. In addition, preliminary studies using fresh bone marrow explants, incubated with Tyrode´s Buffer containing 5% mouse serum, showed an increased migration from the osteoblastic niche to the vascular niche and an enhanced ability to form pro-platelets in C3G transgenic megakaryocytes, as compared to wild-type ones. These results suggest the participation of C3G in different key aspects of megakaryopoiesis by a mechanism mediated by its GEF activity.Peer reviewe

Contribution of the Rap-GEF C3G to signaling pathways in platelets

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.C3G is guanine nucleotide exchange factor for Rap1b, a protein of the Ras family involved in critical aspects of platelet function through the activation of integrin aIIbb3. Using transgenic mouse models, with specific expression in platelets, of full length C3G (tgC3G) or mutant lacking the GEF domain (tgC3GDCat), our group has unveiled an important role of C3G in primary hemostasis. Thus, C3G-Rap1 participates in thrombin-triggered platelet activation, aggregation and clotting through a PKC-mediated pathway. In addition, C3G-overexpressing platelets also show a stronger response to ADP, as compared to wt platelets. Based on these results, we have deepened into the role of C3G in platelet signaling by using several inhibitors of different platelets signaling pathways. This includes: clopidogrel and 2MeSAMP (for P2Y12 ADP receptor), MRS2179 (for P2Y1 ADP receptor), SB203580 (for p38a/b MAPK), U0126 (for ERK1/2), aspirin (for TXA2 synthesis) and wortmannin (for PI3K). We have performed activation, aggregation, and Rap1 activity assays in platelets stimulated with thrombin or ADP/fibrinogen in the presence or absence of the above referred inhibitors. Our results indicate that thrombin- or ADP-stimulated tgC3G platelets are more sensitive to the action of clopidogrel, SB203580, U0126 and aspirin, as compared to wild type platelets. In contrast, tgC3GDCat platelets are more resistant to these inhibitors than their corresponding control platelets. No differences were observed with the rest of inhibitors. These results suggest that C3G may participate in the activation of Rap1 mediated by ADP-P2Y12, but independent of PI3K. Furthermore, our results support a role for C3G in the thromboxane synthesis pathway, probably through modulating ERKs and p38a MAPK activities.Peer reviewe

Mechanisms involved in the regulation of the tumorigenic and invasive properties of colon carcinoma cells by C3G

Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015.C3G is a guanine nucleotide exchange factor (GEF) for some Ras and Rho GTPases, mainly Rap-1. Nevertheless, it also acts through mechanisms independent of its GEF activity. It is essential for embryonic development due to its role on adhesion. C3G regulates several cellular functions such as proliferation, survival and migration. Its function in human cancer is controversial, acting as either a tumour suppressor or promoter depending on the context. p38 MAPKs are activated by several stimuli. They regulate different cellular functions, including migration and invasion. p38(alfa) MAPK is the most abundant isoform, which is essential for embryonic development. It plays a dual role in cancer, acting as a tumour suppressor in initial stages, but promoting migration, invasion and survival at later stages. We have previously identifi ed a functional interaction between C3G and p38a that controls cell death in mouse embryonic fibroblasts and also in chronic myeloid leukemia cells, where C3G-p38a pathway additionally regulates cell adhesion. We have now found that this C3G/p38a cascade also regulates migration and invasion of HCT116 colon carcinoma cells. Thus, C3G knock-down promotes migration/invasion through the enhancement of p38a activation by a Rap-1 independent pathway. We are analyzing if this pro-migratory effect is a consequence of an epithelial to mesenchymal transition (EMT) by measuring different EMT markers. Additionally, our in vitro and in vivo studies have revealed that both C3G and p38a promote tumour growth. Cytokines generated by colon carcinoma cells might indirectly lead to the regulation of tumour growth through its impact on the stroma.Peer reviewe

C3G, a new player in the epithelial-mesenchymal transition in hepatocarcinoma an oval cells

Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015.C3G is a guanine nucleotide exchange factor (GEF) for Rap1 and R-Ras, but it also acts through mechanisms independent of its GEF activity. C3G is essential for embryonic development based on its role in adhesion. Accordingly, it regulates functions related to cytoeskeletal remodelling such as migration and invasion. Its role in cancer is controversial, acting either as a tumour mediator or suppressor. Previously, we have found that C3G knock-down enhanced the migratory and invasive capacity of mouse embryonic fibroblasts and HCT116 colon carcinoma cells through hyperactivation of p38a. Based on this, we aimed to explore if C3G also regulates these processes in hepatocarcinoma (HCC) cells as well as in oval cells, bipotential hepatic progenitors that play an important role in liver regeneration after severe liver damage but have been also involved in HCC development. To do so, we have knocked-down C3G expression in several HCC cell lines displaying different degrees of epithelial phenotype and oval cell lines. Our results indicate that C3G knock-down increased migration and invasion of both HCC and oval cells. Based on the relevance of the epithelial-mesenchymal transition (EMT) for the acquisition of a more migratory and invasive cell phenotype, we are investigating the potential role of C3G on the regulation of EMT under basal conditions and upon induction by TGF-B. Our preliminary results indicate that C3G knock-down promotes E-cadherin loss and controls the expression of the EMT-inducing transcription factors Snail 1, Zeb 1/2 and Twist.Peer reviewe

C3G promotes angiogenesis through the regulation of the differential release of pro-/anti-angiogenic factors from thrombin-activated platelets

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.C3G is a guanine nucleotide exchange factor (GEF) of several proteins within the Ras superfamily, including Rap1a/b and R-Ras. Rap1 is known to be involved in critical aspects of platelet function, including aggregation, adhesion and spreading, through the activation of the platelet integrin αIIbβ3. Using transgenic mouse models expressing C3G or C3GΔCat (a mutant lacking the catalytic domain) in megakaryocytes and platelets, our group has identified C3G as a mediator of Rap1 actions leading to platelet activation and aggregation. Furthermore, C3G promotes α-granule release, evidenced by the increase in P-selectin exposure on the platelet surface, following its activation. In addition, preliminary immunofluorescence analysis suggests that C3G modulates the release of VEGF and endostatin from thrombin-activated platelets. The goal of the present study was to further characterize the underlying mechanisms that regulate the function of C3G in the differential release of pro- and anti-angiogenic factors upon platelet activation, and to evaluate the angiogenic potential of the platelet releasate. We have found that VEGF release was up-regulated in thrombin-activated mouse platelets from both, C3G and C3GΔCat, transgenic mice. Additionally, thrombospondin release was also up-regulated in the latter. The releasate of TgC3G platelets had an overall pro-angiogenic effect as evidenced in a capillary-tube formation assay with HUVECs and in heterotopic tumor mouse models using B16 and 3LL cells. On the other hand, platelet spreading analysis revealed an up-regulation of this function in both transgenic mouse platelets upon thrombin activation, with TgC3G platelets showing the highest extension area. A preliminary co-IP analysis revealed an interaction between C3G and VAMP-7 upon activation, which could explain this result. Taken together, our data indicate the co-existence of RapGEF-dependent and independent mechanism through which C3G is regulating platelet secretion, and suggest an overall pro-angiogenic effect of platelet C3G.Peer reviewe