ABSTRACT
The contribution of endothelial cells to scar and fibrotic tissue formation is undisputedly connected to their ability to undergo the endothelial-to-mesenchymal transition (EndMT) towards fibroblast phenotype-resembling cells. The migration model of fibroblasts and fibroblast-resembling cells is still not fully understood. It may be either a Rho/ROCK-independent, an integrin- and MMP-correlated ECM degradation-dependent, a mesenchymal model or Rho/ROCK-dependent, integrin adhesion- and MMP activity-independent, an amoeboid model. Here, we hypothesized that microvascular endothelial cells (HMEC-1) undergoing EndMT adopt an intermediate state of drifting migration model between the mesenchymal and amoeboid protrusive types in the early stages of fibrosis. We characterized the response of HMEC-1 to TGF-ß2, a well-known mediator of EndMT within the microvasculature. We observed that TGF-ß2 induces up to an intermediate mesenchymal phenotype in HMEC-1. In parallel, MMP-2 is upregulated and is responsible for most proteolytic activity. Interestingly, the migration of HMEC-1 undergoing EndMT is dependent on both ECM degradation and invadosome formation associated with MMP-2 proteolytic activity and Rho/ROCK cytoskeleton contraction. In conclusion, the transition from mesenchymal towards amoeboid movement highlights a molecular plasticity mechanism in endothelial cell migration in skin fibrosis.
Subject(s)
Cell Movement/drug effects , Endothelial Cells/drug effects , Epithelial-Mesenchymal Transition/drug effects , Fibroblasts/drug effects , Matrix Metalloproteinase 2/metabolism , Transforming Growth Factor beta2/pharmacology , Cell Line , Cytoskeleton/drug effects , Cytoskeleton/ultrastructure , Dermis/cytology , Dermis/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Extracellular Matrix/chemistry , Extracellular Matrix/drug effects , Extracellular Matrix/ultrastructure , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Expression Regulation , Humans , Matrix Metalloproteinase 2/genetics , Phenotype , Podosomes/drug effects , Podosomes/metabolism , Podosomes/ultrastructure , Proteolysis , Signal Transduction , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolismABSTRACT
BACKGROUND: The epithelial-mesenchymal transition (EMT) is considered a core process that facilitates the escape of cancer cells from the primary tumor site. The transcription factor Snail was identified as a key regulator of EMT; however, the cascade of regulatory events leading to metastasis remains unknown and new predictive markers of the process are awaited. METHODS: Gene expressions were analysed using real-time PCR, protein level by Western immunoblotting and confocal imaging. The motility of the cells was examined using time-lapse microscopy. Affymetrix GeneChip Human Genome U133 Plus 2.0 analysis was performed to identify transcriptomic changes upon Snail. Snail silencing was performed using siRNA nucleofection. NMU detection was performed by ELISA. RESULTS: HT29 cells overexpressing Snail showed changed morphology, functions and transcriptomic profile indicating EMT induction. Changes in expression of 324 genes previously correlated with cell motility were observed. Neuromedin U was the second highest upregulated gene in HT29-Snail cells. This increase was validated by real-time PCR. Additionally elevated NMU protein was detected by ELISA in cell media. CONCLUSIONS: These results show that Snail in HT29 cells regulates early phenotype conversion towards an intermediate epithelial state. We provided the first evidence that neuromedin U is associated with Snail regulatory function of metastatic induction in colon cancer cells. GENERAL SIGNIFICANCE: We described the global, early transcriptomic changes induced through Snail in HT29 colon cancer cells and suggested NMU involvement in this process.
