RESUMO
CCAAT/enhancer-binding protein-δ (C/EBP-δ), a transcription factor, is elevated in carcinoma compared with that in normal tissue. This study reports a novel function of C/EBP-δ in lymphangiogenesis and tumor metastasis. Genetic deletion of C/EBP-δ in mice resulted in a significant reduction of lymphangiogenesis and pulmonary metastases, with a dramatic reduction of vascular endothelial growth factor-C (VEGF-C) and its cognate receptor VEGF receptor-3 (VEGFR3) in lymphatic endothelial cells (LECs). By contrast, no difference of VEGF-C in tumor tissues and bone marrow was observed between null and wild-type mice. Consistently, forced expression of C/EBP-δ increased VEGF-C and VEGFR3 expression in cultured LECs. These findings suggest a specific and important role of C/EBP-δ in the regulation of VEGFR3 signaling in LECs. Furthermore, expression of C/EBP-δ in cultured LECs significantly increased cell motility, and knockdown of C/EBP-δ inhibited cell motility and lymphatic vascular network formation in vitro. Forced expression of VEGF-C, but not recombinant VEGF-C, rescued the knockdown of C/EBP-δ-induced cell apoptosis, indicative of autonomous VEGF-C autocrine signaling essential for LEC survival. Moreover, hypoxia induces C/EBP-δ expression and C/EBP-δ regulates HIF-1α expression. Blocking HIF-1α activity totally blocked CEBP-δ-induced VEGF-C and VEGFR3 expression in LECs. Together, these findings uncover a new function of CEBP-δ in lymphangiogenesis through regulation of VEGFR3 signaling in LECs.
Assuntos
Comunicação Autócrina , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Linfangiogênese , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Proteína delta de Ligação ao Facilitador CCAAT/deficiência , Proteína delta de Ligação ao Facilitador CCAAT/genética , Hipóxia Celular , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/genética , Camundongos , Metástase Neoplásica , Especificidade por Substrato , Fator C de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The objective of this study was to develop a test method for determining the cytotoxicity and biocompatibility of various biomaterials that are used in orthopaedic surgery. This method is based on the use of a human bone marrow cell culture and was developed as an alternative to animal experiments. Human bone marrow cell culture has certain advantages over other cell culture models, as its results show a greater conformity with animal experimental results and clinical studies. Primary cell adherence, cell number, cell proliferation, production of extracellular matrix, cell viability and cell differentiation were used as indicative parameters of biocompatibility. After 2 weeks in culture, differences could be observed between the biomaterials with respect to these parameters. Cell numbers were greatest on the hydroxyapatite ceramic specimens, but were decreased on the titanium alloy specimens. Extracellular matrix hydroxyapatite production was high for ceramics, but reduced for titanium specimens. The polymers allowed only a few cells to adhere, and there were no signs of extracellular matrix production. The influence of biomaterials on differentiation of large numbers of cells was analysed by using flow cytophotometry. There were similar populations of T cells and monocytes on all specimens. However, extended B cell and granulocyte populations were observed with titanium and polyethylene.
