Résumé
Objective To investigate the effects of rotavirus ( RV) on the expression and bioactiv-ity of Na+-H+ exchanger 3 ( NHE3 ) in Caco-2 cells and the possible regulatory mechanism. Methods Caco-2 cells expressing NHE3 were constructed and divided into four groups as follows: control ( CTL ) group, RV group, BAPTA-AM ( a Ca2+ chelator) group and BAPTA-AM+RV group. Na+-H+ exchanger ac-tivity and NHE3 expression on cell surface were determined using BCECF-AM and biotinylation assay, re-spectively. Expression of Cdc42 at protein level was measured by Western blot. Results Compared with the control group, RV infection significantly decreased the activity of NHE3 and its expression on cell surface. BATPA-AM antagonized the inhibitory effects on NHE3. Moreover, the expression of Cdc42 at protein level was increased following RV infection, which was also antagonized by BATPA-AM. Conclusions Intracellu-lar Ca2+-mediated Cdc42-dependent endocytosis pathway might be involved in regulating the expression and bioactivity of NHE3 during RV infection.
Résumé
Objective To observe the effects and regulatory mechanism of rotavirus infection on the expression and bioactivity of Na+/H+exchanger 3 (NHE3) on Caco-2 cells. Methods A cell model of Caco-2 cells expressing NHE3 was constructed. Four groups were set up,which were control(CTL) group, rotavirus(RV) infection group, Cdc42 inhibitor (Pirl-1) group and Pirl-1+RV group. Bioactivity and ex-pression of NHE3 on the surface of Caco-2 cells were determined by BCECF-AM and biotinylation method, respectively. Expression of Cdc42 protein was measured by Western blot. Co-immunoprecipitation was per-formed to detect the interaction between NHE3 and Cdc42. Results Compared with the CTL group,RV in-fection significantly inhibited the bioactivity and expression of NHE3 on Caco-2 cells. These inhibitory effects were antagonized by Pirl-1. Moreover,RV infection enhanced the expression of Cdc42 protein and promoted the interaction between NHE3 and Cdc42, which were also antagonized by Pirl-1. Conclusion RV infec-tion might regulate the expression and bioactivity of NHE3 through Cdc42-dependent endocytosis pathway.
Résumé
Objective To observe the effect of rotavirus (RV) infection on expression level and bioactivity of Na+-H+ exchanger 3 (NHE3) in Caco-2 cells. Methods Model of NHE3-expressing Caco-2 cells was constructed and studied in terms of intervention: control, RV, clathrin antagonist chlorpromazine (CPZ), and CPZ + RV. NHE3 activity and NHE3 protein amount on cell surface were determined by BCECF-AM and biotinylation, respectively. Expression level of clathrin was assayed by Western blot. Results Compared with control group, NHE3 activity and NHE3 surface protein level significantly decreased when the cells were treated with RV. These effects could not be completely cancelled by clathrin antagonist CPZ. Moreover, RV treatment could increase cellular protein level of clathrin, which was cancelled by CPZ. Conclusions The effect of RV infection on NHE3 expression level and biological activity may be related to clathrin-dependent endocytosis pathway, and may be also affected by other endocytosis pathways.
Résumé
In this study, the endocytosis pathway of heparosan and its intracellular distribution were investigated in MCF-7 tumor cells and COS7 normal cells. The endocytosis inhibition and cellular probe location experiments showed that MCF-7 tumor cells took heparosan more efficiently and selectively than COS7 cells. The cellular uptake of heparosan was energy-dependent in both MCF-7 tumor cells and COS7 normal cells. Moreover, the major endocytosis pathway of heparosan into MCF-7 tumor cells was caveolin-mediated endocytosis and macropinocytosis. The internalized heparosan was mainly located in lysosomes of the cells.