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

Abstract

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 C3G∆Cat 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.

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

“…The production of megakaryocytes from adult HSC of bone marrow (BM), cultured in the presence of TPO and a cocktail of cytokines, is an ex vivo model commonly used to study the mechanisms involved in the expansion and differentiation of megakaryocytic progenitors. To study, in this ex vivo model, the participation of C3G in megakaryopoiesis, we have used two lineages of transgenic mice for C3G (2C1 and 6A6) and one for C3GΔCat (8A3 lineage), where the transgenes are expressed under the PF4 gene promoter [21, 23].…”

“…In addition, we have recently described a role of C3G in platelet secretion, mainly through GEF-independent mechanisms. Thus, transgenic expression of C3G in platelets modulates α-granule exocytosis through interaction with VAMP-7, which results in an overall proangiogenic secretome, facilitating inflammatory angiogenesis and tumor metastasis [23].…”

How Rap and its GEFs control liver physiology and cancer development. C3G alterations in human hepatocarcinoma

“…C3G expression increases in human non-small-cell lung cancer [ 38 ] and in the papillary thyroid carcinoma, C3G is also implicated [ 39 ]. In addition, transgenic expression of C3G in platelets promotes tumor angiogenesis and metastasis [ 40 ]. Finally, chronic myeloid leukemia is associated with a high expression of the p87C3G isoform [ 17 ].…”

C3G contributes to platelet activation and aggregation by regulating major signaling pathways

“…The transgenic mice models used in this work have been previously described. [2][3][4] The human transgenes C3G (full-length) and C3GΔCat (deletion in the catalytic region) were expressed under the control of the platelet-specific PF4 gene promoter. Transgenic C3G (tgC3G) lines 2C1 and 6A6 and transgenic C3GΔCat (tgC3GΔCat) line 8A3 were used.…”

“…Transgenic expression of C3G in platelets stimulates (i) megakaryocytic differentiation and proplatelet formation, 2 (ii) platelet activation and aggregation, 3 and (iii) αgranule secretion and platelet angiogenesis and metastasis. 4 In addition, we have shown that C3G participates in the activation of Rap1 triggered by thrombin and PMA (phorbol 12-myristate 13acetate) through the PKC pathway. 3 Other investigations have demonstrated the formation of a ternary VASP/CrkL/C3G complex that activates platelet Rap1b.…”