RESUMO
We have generated mouse transgenic lineages for C3G (tgC3G) and C3GΔCat (tgC3GΔCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GΔCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GΔCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.
Assuntos
Plaquetas/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/genética , Ativação Plaquetária , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Células Cultivadas , Ativação Enzimática , Feminino , Engenharia Genética , Fator 2 de Liberação do Nucleotídeo Guanina/biossíntese , Humanos , Contagem de Leucócitos , Masculino , Megacariócitos/metabolismo , Megacariócitos/fisiologia , Camundongos , Camundongos Transgênicos , Neutrófilos/fisiologia , Contagem de Plaquetas , Fator Plaquetário 4/genética , Regiões Promotoras Genéticas , Proteína Quinase C/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
The Ras-CRK-Rap1 cellular signal-transduction system is regulated by guanine nucleotide exchange factors (GEFs). Transcription of C3G on chromosome 9q34 and a key member of the GEF gene family is activated by the CRK-adaptor protein; the C3G product is a CRK SH3 domain-binding guanine nucleotide-releasing factor. We document here the amplification of C3G in five of 18 primary non-small cell lung cancers examined and its increased expression in 18 of 28 tumors in comparison to corresponding non-cancerous lung tissues. Immunohistochemical staining revealed prominent C3G protein in the cytoplasm of cancer cells, associated with faint staining at the nucleolar membrane, but C3G was not detectable in normal bronchial mucoepithelial cells or in broncholoalveolar cells of the bronchial/bronchiolar ducts or alveoli. These data indicate that amplification and increased expression of the C3G gene may play some role in human lung carcinogenesis through derangement of the CRK-Rap1 signaling pathway.
