Abstract
Voltage-gated sodium channels initiate electrical signaling in excitable cells such as muscle and neurons. They also are expressed in non-excitable cells such as macrophages and neoplastic cells. Previously, in macrophages, we demonstrated expression of SCN8A, the gene that encodes the channel NaV1.6, and intracellular localization of NaV1.6 to regions near F-actin bundles, particularly at areas of cell attachment. Here we show that a splice variant of NaV1.6 regulates cellular invasion through its effects on podosome and invadopodia formation in macrophages and melanoma cells. cDNA sequence analysis of SCN8A from THP-1 cells, a human monocyte-macrophage cell line, confirmed the expression of a full-length splice variant that lacks exon 18. Immunoelectron microscopy demonstrated NaV1.6-positive staining within the electron dense podosome rosette structure. Pharmacologic antagonism with tetrodotoxin (TTX) in differentiated THP-1 cells or absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation. Agonist-mediated activation of the channel with veratridine caused release of sodium from cationic vesicular compartments, uptake by mitochondria, and mitochondrial calcium release through the Na/Ca exchanger. Invasion by differentiated THP-1 and HTB-66 cells, an invasive melanoma cell line, through extracellular matrix was inhibited by TTX. THP-1 invasion also was inhibited by small hairpin RNA knockdown of SCN8A. These results demonstrate that a variant of NaV1.6 participates in the control of podosome and invadopodia formation and suggest that intracellular sodium release mediated by NaV1.6 may regulate cellular invasion of macrophages and melanoma cells.
Highlights
In excitable tissues such as muscle and nerve, activation of voltage-gated sodium channels initiates electrical signaling through sodium influx coupled to membrane depolarization [1]
These findings suggest that a variant of NaV1.6 contributes to the control of macrophage and melanoma cellular invasion through a signaling pathway that may link intracellular sodium channel activation to actin cytoskeleton dynamics
In primary mouse peritoanoma Invadopodia and Macrophage Podosomes—Previously neal macrophages treated with a chemotactic stimulus we demonstrated by immunofluorescence and immuno-EM, NaV1.6-positive vesicles were closely that macrophage NaV1.6 appeared to localize to F-actin fiber associated with gelsolin, a calcium-dependent regulator of actin bundles [6]
Summary
Cells—THP-1 cells, a human premyelomonocytic leukemic cell line, were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, and nonessential amino acids. Podosome Quantitation—Differentiated THP-1 cells were treated with serum-free media for 4 h in the presence and absence of 300 nM TTX, a concentration that blocks TTX-sensitive sodium channels such as NaV1.6 but not TTX-resistant channels such as NaV1.5. They were permeabilized in PBS containing 0.1% Triton X-100 for 20 min, incubated with phalloidin-Alexa 488 in 1% bovine serum albumin for an additional 20 min, and washed with PBS. Fluorometry—For time-resolved fluorescence analysis of THP-1 cells or freshly isolated mouse peritoneal macrophages, cells were isolated by centrifugation, resuspended in serumfree HBSS at room temperature, and labeled with indicator dyes. Image Processing—Microscopic images were obtained using Axiovision software as described above, exported as TIFF files, compiled in composite figures using Adobe Illustrator, and exported as TIFF files
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