ASIC proteins regulate smooth muscle cell migration

Abstract

The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated that Epithelial Na(+)Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration; however, the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence individual ASIC expression and determine the importance of ASIC proteins in wound healing and chemotaxis (PDGF-bb)-initiated migration. We found ASIC1, ASIC2, and ASIC3, but not ASIC4, expression in A10 cells. ASIC1, ASIC2, and ASIC3 siRNA molecules significantly suppressed expression of their respective proteins compared to non-targeting siRNA (RISC) transfected controls by 63%, 44%, and 55%, respectively. Wound healing was inhibited by 10, 20, and 26% compared to RISC controls following suppression of ASIC1, ASIC2, and ASIC3, respectively. Chemotactic migration was inhibited by 30% and 45%, respectively, following suppression of ASIC1 and ASIC3. ASIC2 suppression produced a small, but significant, increase in chemotactic migration (4%). Our data indicate that ASIC expression is required for normal migration and may suggest a novel role for ASIC proteins in cellular migration.

Figure 1. ASIC message expression in cultured VSMCs

Representative images of RT-PCR detection of ASIC1a, ASIC2a, and ASIC3, but not ASIC4 transcripts. The presence (RT+) and absence (RT−) of reverse transcriptase is indicated. Brain was used as positive control.

Figure 2. ASIC protein expression by immunolabeling in cultured VSMC

Representative images of ASIC1a, ASIC2a, and ASIC3 protein expression in cultured VSMC. Antibody labeling is blocked when the antibody is incubated with excess antigen. α-actin or golgi stain (NBD-ceramide) was used as a positive control. Punctate cytoplasmic and perinuclear staining were typically observed.

Figure 3. ASIC protein expression by western blotting in cultured VSMC

Western blot detection of ASIC1a, ASIC2a, and ASIC3 protein in whole cell VSMC lysates. Antibody labeling is blocked when the antibody is incubated with excess antigen. β-Actin loading control is shown below the corresponding ASIC blot.

Figure 4. Co-localization of ASIC proteins in VSMCs

Representative images shown in A–C and quantitative data shown in D. A–C. Co-labeling of ASIC proteins, as indicated, is shown in the first two panels. A merged image is shown in the third panel. Yellow coloration indicates co-localization. An enlarged image of a cytoplasmic region in the same cell is shown in the far right panel. D. Co-localization of ASIC1/ASIC2, ASIC1/ASIC3 and ASIC2/ASIC3 is approximately 20% in the perinuclear regions, but only 1–2% in cytoplasmic regions.

Figure 5. Silencing ASIC expression by siRNA approach inhibits ASIC protein expression in VSMC in vitro

A. Quantitative effect of siRNA on ASIC expression. Delivery of individual ASIC1, ASIC2 or ASIC3 siRNA (100 nM) reduces the respective ASIC protein expression in cultured VSMCs when compared to non-targeting siRNA (RISC) controls. B. Representative images of ASIC1 and α-actin immunostaining in cultured VSMCs following transfection with ASIC1 or RISC (negative control) siRNA molecules. Data are mean ± SEM. * Significantly different from respective control, p<0.05.

Figure 6. Silencing ASIC expression inhibits VSMC migration in vitro

A and B. ASIC1, ASIC2 or ASIC3 silencing on wound healing at 8 and 24 hours. At 8 hours, only ASIC3 suppression significantly inhibits wound healing. However, by 24 hours, suppression of ASIC1, ASIC2 or ASIC3 expression inhibits wound healing. C. Suppression of ASIC1 and ASIC3 inhibits migration in response to PDGF-bb, while ASIC2 suppression enhances migration. D. Random migration responses were unaltered by ASIC silencing. Data are mean ± SEM. * Significantly different from control, p<0.05.

Figure 7. Silencing ASIC expression does not affect VSMC adhesion in vitro

VSMC adhesion to fibronectin following ASIC silencing was not different from RISC control. Data are mean ± SEM. * Significantly different from RISC control, p<0.05.