Ionomycin ameliorates hypophosphatasia via rescuing alkaline phosphatase deficiency-mediated L-type Ca2+ channel internalization in mesenchymal stem cells

摘要

ALPL deficiency caused decreased membrane expression of L-type Ca channels in BMSCs. Ca imaging showed decreased Ca influx in cultured BMSCs and WT BMSCs transfected with shALP (shALP/WT) after they were stimulated with 30 mmol·L KCl for 3 min (  = 10). No KCl-induced Ca influx was detected in cultured WT, , and shALP/WT BMSCs treated with 10 mmol·L EGTA for 3 min (  = 10). ALPL overexpression was mediated by a lentivirus in (Lenti-alp/ ) BMSCs and resulted in an elevated Ca influx following stimulation with 30 mmol·L KCl for 3 min (  = 10). , The expression of Ca 1.1, Ca 1.2, and Ca 1.3 was assessed. BMSCs showed decreases in total cell expression ( ) and membrane expression of Ca 1.2 and Ca 1.3 ( ) and no significant change in the levels of cytoplasmic Ca 1.2 and Ca 1.3 ( ). Total Ca 1.1 protein expression was not changed ( ), and the expression of membrane and cytoplasmic Ca 1.1 was not altered in BMSCs ( ). Cell-surface biotinylation assay. Left two lanes: western blot for Ca 1.2 and Ca 1.3 following neutravidin pull down from WT and BMSCs; right two lanes: input, not biotinylated cells. Lenti-alp/ BMSCs showed elevated membrane expression of ALP, Ca 1.2, and Ca 1.3. , Representative images of confocal laser scanning microscopy showing the membrane location of Ca 1.2 and Ca 1.3 (green) in WT and Lenti-alp/ BMSCs. The plasma membrane was stained with the marker CellMask™ Deep Red Plasma Membrane Stain (red) ( ). Quantification of the membrane florescence was performed with NIH ImageJ ( ). Scale bar, 10 μm. The representative results from three independent experiments are shown. Error bars represent the s.d. from the mean values. *  P P