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Abstract
BackgroundBinding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) makes neutrophils roll on and adhere to inflammatory site. Intracellular calcium bursting of adhered neutrophils is a key event for subsequent arresting firmly at and migrating into the injured tissue. But, it remains unclear how the cytoplasmic calcium signaling of the cells were modulated by the fluid shear stress. Here, we focus on mechanical regulation of P-selectin-induced calcium signaling of neutrophil-like HL-60 cells under flow.MethodsHL-60 cells were loaded with Fluo-4 AM for fluorescent detection of intracellular calcium ion, and then perfused over P-selectin-coated bottom of parallel-plate flow chamber. The intracellular calcium concentration of firmly adhered cell under flow was observed in real time by fluorescence microscopy.ResultsForce triggered, enhanced and quickened cytoplasmic calcium bursting of HL-60 on P-selectin. This force-dependent calcium signaling was induced by the immobilized P-selectin coated on substrates in absence of chemokine. Increasing of both shear stress and P-selectin concentration made the calcium signaling intensive, through quickening the cytosolic calcium release and upregulating both probability and peak level of calcium signaling.ConclusionsImmobilized P-selectin-induced calcium signaling of HL-60 cells is P-selectin concentration- and mechanical force-dependent. The higher both the P-selectin concentration and the external force on cell, the more intensive the calcium signaling. It might provide a novel insight into the mechano-chemical regulation mechanism for intracellular signaling pathways induced by adhesion molecules.
Highlights
Binding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) makes neutrophils roll on and adhere to inflammatory site
P‐selectin‐induced calcium signaling in HL‐60 cells was specific and concentration‐dependent We examined the calcium signaling of HL-60 cells on blank and P-selectin (0.1, 1 and 10 μg/mL with BSA)-coated substrates, and measured the cell activation ratio, which was defined as the percentage of calcium signaling cells in all firmly adhered cells under the field of view, to estimate the probability of calcium signaling
Increasing of wall shear stress would upregulate the cell activation ratio or the probability of calcium signaling of HL-60 (Fig. 4b) and rise the peak level of calcium signaling (Fig. 4c) and quicken the cytosolic calcium release of HL-60 (Fig. 4d). These results revealed that mechanical force, as a trigger and regulator for P-selectininduced calcium signaling of HL-60, was required for significant increment of cytosolic ionized calcium
Summary
Proteins and cells Human promyelocytic leukemia HL-60 cells (Cell Bank of Chinese Academy of Sciences, Shanghai, China), constitutively expressed PSGL-1 as a ligand for P-selectin, were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 10 mg/ mL streptomycin and 100 units/mL penicillin at 37 °C in a humidified atmosphere of 5% CO2 in air. After removal of excessive unabsorbed P-selectin, polystyrene petri dishes were washed with PBS containing 1% BSA 3 times, and incubated in the same solution for 1 h at room temperature to block nonspecific cell adhesion. The site densities of 0.1, 1.0 and 10 μg/ mL P-selectin absorbed on polystyrene petri dish were determined to be 21, 208 and 1359 #/μm. The site densities of 0.1, 1.0 and 10 μg/ mL P-selectin absorbed on polystyrene petri dish were determined to be 21, 208 and 1359 #/μm2 These P-selectin densities were selected to support the firm adhesion of HL-60 on the substrates. 1 μM Fluo-4 AM was loaded into cells by incubating cells at a concentration of 1 × 106/mL for 30 min at 37 °C in loading buffer (20 mM HEPES, 20 mM Glucose and 1% BSA in PBS). Statistics methods The data were compared by one-way analysis of variance (ANOVA) and Student’s t test, and considered statistically significant if p < 0.05
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