ABSTRACT
Lymphangiogenesis plays a crucial role during development, in cancer metastasis and in inflammation. Activation of VEGFR-3 (also known as FLT4) by VEGF-C is one of the main drivers of lymphangiogenesis, but the transcriptional events downstream of VEGFR-3 activation are largely unknown. Recently, we identified a wave of immediate early transcription factors that are upregulated in human lymphatic endothelial cells (LECs) within the first 30 to 80â min after VEGFR-3 activation. Expression of these transcription factors must be regulated by additional pre-existing transcription factors that are rapidly activated by VEGFR-3 signaling. Using transcription factor activity analysis, we identified the homeobox transcription factor HOXD10 to be specifically activated at early time points after VEGFR-3 stimulation, and to regulate expression of immediate early transcription factors, including NR4A1. Gain- and loss-of-function studies revealed that HOXD10 is involved in LECs migration and formation of cord-like structures. Furthermore, HOXD10 regulates expression of VE-cadherin, claudin-5 and NOS3 (also known as e-NOS), and promotes lymphatic endothelial permeability. Taken together, these results reveal an important and unanticipated role of HOXD10 in the regulation of VEGFR-3 signaling in lymphatic endothelial cells, and in the control of lymphangiogenesis and permeability.
Subject(s)
Homeodomain Proteins/genetics , Neoplasms/genetics , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Transcription Factors/genetics , Vascular Endothelial Growth Factor C/genetics , Vascular Endothelial Growth Factor Receptor-3/genetics , Cell Line , Cell Membrane Permeability/genetics , Cell Movement/genetics , Endothelial Cells/metabolism , Endothelial Cells/pathology , Gene Expression Regulation, Neoplastic , Humans , Lymphangiogenesis/genetics , Neoplasm Metastasis , Neoplasms/pathology , Signal Transduction , Vascular Endothelial Growth Factor C/biosynthesis , Vascular Endothelial Growth Factor Receptor-3/biosynthesisABSTRACT
VEGF-C/VEGFR-3 signaling plays a central role in lymphatic development, regulating the budding of lymphatic progenitor cells from embryonic veins and maintaining the expression of PROX1 during later developmental stages. However, how VEGFR-3 activation translates into target gene expression is still not completely understood. We used cap analysis of gene expression (CAGE) RNA sequencing to characterize the transcriptional changes invoked by VEGF-C in LECs and to identify the transcription factors (TFs) involved. We found that MAFB, a TF involved in differentiation of various cell types, is rapidly induced and activated by VEGF-C. MAFB induced expression of PROX1 as well as other TFs and markers of differentiated LECs, indicating a role in the maintenance of the mature LEC phenotype. Correspondingly, E14.5 Mafb(-/-) embryos showed impaired lymphatic patterning in the skin. This suggests that MAFB is an important TF involved in lymphangiogenesis.
Subject(s)
Lymphangiogenesis , MafB Transcription Factor/physiology , Transcriptome , Animals , Antigens, Differentiation/metabolism , Base Sequence , Binding Sites , Cell Differentiation , Cells, Cultured , Embryonic Development , Endothelium, Lymphatic/metabolism , Gene Expression Profiling , Humans , Lymphatic Vessels/cytology , Lymphatic Vessels/metabolism , Male , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Transcriptional Activation , Vascular Endothelial Growth Factor Receptor-3/metabolismABSTRACT
The important role of the lymphatic vascular system in pathological conditions such as inflammation and cancer has been increasingly recognized, but its potential as a pharmacological target is poorly exploited. Our study aimed at the identification and molecular characterization of lymphatic-specific G protein-coupled receptors (GPCRs) to assess new targets for pharmacological manipulation of the lymphatic vascular system. We used a TaqMan quantitative RT-PCR-based low density array to determine the GPCR expression profiles of ex vivo isolated intestinal mouse lymphatic (LECs) and blood vascular endothelial cells (BECs). GPR97, an orphan adhesion GPCR of unknown function, was the most highly and specifically expressed GPCR in mouse lymphatic endothelium. Using siRNA silencing, we found that GPR97-deficient primary human LECs displayed increased adhesion and collective cell migration, whereas single cell migration was decreased as compared with nontargeting siRNA-transfected control LECs. Loss of GPR97 shifted the ratio of active Cdc42 and RhoA and initiated cytoskeletal rearrangements, including F-actin redistribution, paxillin and PAK4 phosphorylation, and ß1-integrin activation. Our data suggest a possible role of GPR97 in lymphatic remodeling and furthermore provide the first insights into the biological functions of GPR97.
Subject(s)
Cell Movement , Endothelial Cells/cytology , Receptors, G-Protein-Coupled/metabolism , cdc42 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Cell Adhesion , Endothelial Cells/enzymology , Endothelial Cells/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Gene Silencing , Humans , Integrin beta1/metabolism , Intestines/cytology , Mice , Paxillin/metabolism , Phosphorylation , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , p21-Activated Kinases/metabolismABSTRACT
Vascular endothelial growth factor A (VEGFA) plays a key role in the angiogenesis of human skin. Elevated levels of VEGFA are associated with several pathological conditions, including chronic inflammatory skin diseases and several types of skin cancer. In particular, squamous cell carcinoma (SCC) of the skin, the second most common skin cancer in the general population, is characterized by invasive growth, pronounced angiogenesis and elevated levels of VEGFA. The processing, turnover and production of VEGFA are extensively regulated at the post-transcriptional level, both by RNA-binding proteins and microRNAs (miRNAs). In the present study, we identified a new miRNA recognition element in a downstream conserved region of the VEGFA 3'-UTR. We confirmed the repressive effect of miR-361-5p on this element in vitro, identifying the first target for this miRNA. Importantly, we found that miR-361-5p levels are inversely correlated with VEGFA expression in SCC and in healthy skin, indicating that miR-361-5p could play a role in cancers.
Subject(s)
Carcinoma, Squamous Cell/genetics , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Skin Neoplasms/genetics , Vascular Endothelial Growth Factor A/genetics , 3' Untranslated Regions , Base Sequence , Carcinoma, Squamous Cell/metabolism , Cell Line , Gene Order , Humans , MicroRNAs/metabolism , Mutation , Skin/metabolism , Skin Neoplasms/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Lipid-bilayer permeation is determinant for the disposition of xenobiotics in the body. It controls the pharmacokinetic behavior of drugs and is, in many cases, a prerequisite for intracellular targeting. Permeation of in vivo barriers is in general predicted from lipophilicity and related parameters. This article goes beyond the empirical correlations, and elucidates the processes and their interplay determining bilayer permeation. A flip-flop model for bilayer permeation, which considers the partitioning rate constants beside the translocation rate constants, is compared with the diffusion model based on Fick's first law. According to the flip-flop model, the ratios of aqueous volumes to barrier area can determine whether partitioning or translocation is rate-limiting. The flip-flop model allows permeation of anions and cations, and expands our understanding of pH-dependent permeation kinetics. Some experimental evidences for ion-controlled permeation at pH 7 are also included in this work.
