Urea‐modulated UT‐B urea transporter internalization is clathrin‐ and caveolae‐dependent in infantile hemangioma‐derived vascular endothelial cells

Abstract

The aim of this study was to investigate the manner of urea‐modulated UT‐B urea transporter (UT) internalization in infantile hemangioma‐derived vascular endothelial cells (HemECs). The immunohistochemistry assay was performed to identify infancy hemangioma‐derived endothelial cell line (XPTS‐1) cells. Cell toxicity was detected with the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide (MTT) assay. Quantitative real‐time polymerase chain reaction and Western blot analysis were measured to analyze the expression of UT‐B. UT‐B internalization was observed by confocal microscopy. The clathrin inhibitor chlorpromazine (CPZ) and caveolin endocytic disrupter methyl‐β‐cyclodextrin (MβCD) were used in XPTS‐1 cells transfected with UT‐B‐GFP to repress endocytosis. Urea‐promoted UT‐B expression in a concentration‐dependent manner in an infantile XPTS‐1 cell line. CPZ and MβCD significantly inhibited UT‐B protein internalization. The pretreatment of UT‐B‐GFP cells with adaptor protein2 (AP2)‐μ2‐siRNA and caveolin‐siRNA significantly inhibited UT‐B protein internalization. Our findings suggested that urea‐mediated UT‐B UT internalization is clathrin and caveolae dependent in infantile HemECs.

Toll-Like Receptor-Mediated Activation of CD39 Internalization in BMDCs Leads to Extracellular ATP Accumulation and Facilitates P2X7 Receptor Activation

“…To test this hypothesis, experiments involving the addition of endocytosis inhibitors were performed. Many endocytosis inhibitors, including selective and non-selective inhibitors of clathrin-dependent and clathrin-independent pathways of endocytosis (CDE and CIE, respectively), were tested in the experiment (46–48). The downregulation of membrane CD39 expression in LPS-treated BMDCs was almost completely blocked by the addition of Dynasore, a non-selective endocytosis inhibitor (Figure 2A).…”

Enterovirus 71 enters human brain microvascular endothelial cells through an ARF6‐mediated endocytic pathway

“…17 It is reported that the uptake of transferrin could be used as an effective indicator of clathrin-mediated endocytosis. 18 In this study, CPZ reduced the uptake of AF488-bound transferrin into HBMECs (Figure 2A), while CPZ did not reduce the rate of EV71 infection (Figure 2B). These data suggested that EV71 infection occurred in a manner that was independent of clathrin-mediated endocytic pathway.…”

“…Chlorpromazine (CPZ), a clathrin‐specific inhibitor, was used to test whether EV71 entry occurred via the clathrin‐related endocytic pathway 17 . It is reported that the uptake of transferrin could be used as an effective indicator of clathrin‐mediated endocytosis 18 . In this study, CPZ reduced the uptake of AF488‐bound transferrin into HBMECs (Figure 2A), while CPZ did not reduce the rate of EV71 infection (Figure 2B).…”