ABSTRACT
Epithelial cell migration is crucial for the development and regeneration of epithelial tissues. Aberrant regulation of epithelial cell migration has a major role in pathological processes such as the development of cancer metastasis and tissue fibrosis. Here, we report that in response to factors that promote cell motility, the Rap guanine exchange factor RAPGEF2 is rapidly phosphorylated by I-kappa-B-kinase-ß and casein kinase-1α and consequently degraded by the proteasome via the SCF(ßTrCP) ubiquitin ligase. Failure to degrade RAPGEF2 in epithelial cells results in sustained activity of Rap1 and inhibition of cell migration induced by HGF, a potent metastatic factor. Furthermore, expression of a degradation-resistant RAPGEF2 mutant greatly suppresses dissemination and metastasis of human breast cancer cells. These findings reveal a molecular mechanism regulating migration and invasion of epithelial cells and establish a key direct link between IKKß and cell motility controlled by Rap-integrin signaling.
Subject(s)
Casein Kinase Ialpha/metabolism , Cell Movement/physiology , Epithelial Cells/cytology , Guanine Nucleotide Exchange Factors/metabolism , I-kappa B Kinase/metabolism , Nerve Tissue Proteins/metabolism , Signal Transduction/physiology , Zebrafish Proteins/metabolism , Animals , Animals, Genetically Modified , Breast Neoplasms , Cell Line, Tumor , Female , HEK293 Cells , Heterografts , Humans , Male , Phosphorylation/physiology , SKP Cullin F-Box Protein Ligases/metabolism , ZebrafishABSTRACT
Lymphatic vessels are derived from venous endothelial cells and their formation is governed by the Vascular endothelial growth factor C (VegfC)/Vegf receptor 3 (Vegfr3; Flt4) signaling pathway. Recent studies show that Collagen and Calcium Binding EGF domains 1 protein (Ccbe1) enhances VegfC-dependent lymphangiogenesis. Both Ccbe1 and Flt4 have been shown to be indispensable for lymphangiogenesis. However, how these essential players are transcriptionally regulated remains poorly understood. In the case of angiogenesis, atypical E2fs (E2f7 and E2f8) however have been recently shown to function as transcriptional activators for VegfA. Using a genome-wide approach we here identified both CCBE1 and FLT4 as direct targets of atypical E2Fs. E2F7/8 directly bind and stimulate the CCBE1 promoter, while recruitment of E2F7/8 inhibits the FLT4 promoter. Importantly, inactivation of e2f7/8 in zebrafish impaired venous sprouting and lymphangiogenesis with reduced ccbe1 expression and increased flt4 expression. Remarkably, over-expression of e2f7/8 rescued Ccbe1- and Flt4-dependent lymphangiogenesis phenotypes. Together these results identified E2f7/8 as novel in vivo transcriptional regulators of Ccbe1 and Flt4, both essential genes for venous sprouting and lymphangiogenesis.
Subject(s)
Calcium-Binding Proteins/metabolism , Lymphangiogenesis/physiology , Vascular Endothelial Growth Factor Receptor-3/metabolism , Zebrafish Proteins/metabolism , Animals , Blotting, Western , Calcium-Binding Proteins/genetics , Chromatin Immunoprecipitation , Electrophoresis, Polyacrylamide Gel , Humans , Lymphangiogenesis/genetics , Vascular Endothelial Growth Factor Receptor-3/genetics , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
Recently, we showed that E2F7 and E2F8 (E2F7/8) are critical regulators of angiogenesis through transcriptional control of VEGFA in cooperation with HIF. (1) Here we investigate the existence of other novel putative angiogenic E2F7/8-HIF targets, and discuss the role of the RB-E2F pathway in regulating angiogenesis during embryonic and tumor development.
Subject(s)
E2F Transcription Factors/metabolism , Hypoxia-Inducible Factor 1/metabolism , Repressor Proteins/metabolism , Animals , Binding Sites , E2F Transcription Factors/deficiency , E2F Transcription Factors/genetics , Humans , Neoplasms/metabolism , Neoplasms/pathology , Neovascularization, Pathologic , Receptors, Vascular Endothelial Growth Factor/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The E2F family of transcription factors plays an important role in controlling cell-cycle progression. While this is their best-known function, we report here novel functions for the newest members of the E2F family, E2F7 and E2F8 (E2F7/8). We show that simultaneous deletion of E2F7/8 in zebrafish and mice leads to severe vascular defects during embryonic development. Using a panel of transgenic zebrafish with fluorescent-labelled blood vessels, we demonstrate that E2F7/8 are essential for proper formation of blood vessels. Despite their classification as transcriptional repressors, we provide evidence for a molecular mechanism through which E2F7/8 activate the transcription of the vascular endothelial growth factor A (VEGFA), a key factor in guiding angiogenesis. We show that E2F7/8 directly bind and stimulate the VEGFA promoter independent of canonical E2F binding elements. Instead, E2F7/8 form a transcriptional complex with the hypoxia inducible factor 1 (HIF1) to stimulate VEGFA promoter activity. These results uncover an unexpected link between E2F7/8 and the HIF1-VEGFA pathway providing a molecular mechanism by which E2F7/8 control angiogenesis.
