Regulation of podosome formation in macrophages by a splice variant of the sodium channel SCN8A.
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.
Carrithers
Michael D
MD
Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06520-8018, USA. carrithers@neurology.wisc.edu
Chatterjee
Gouri
G
Carrithers
Lisette M
LM
Offoha
Roosevelt
R
Iheagwara
Uzoma
U
Rahner
Christoph
C
Graham
Morven
M
Waxman
Stephen G
SG
D017398:Alternative Splicing; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D021961:Cell Membrane Structures / Q000378:metabolism*; D005091:Exons; D006801:Humans; D017737:Macrophages, Peritoneal / Q000378:metabolism* / Q000473:pathology; D008545:Melanoma / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008817:Mice, Mutant Strains; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D062557:NAV1.6 Voltage-Gated Sodium Channel; D009361:Neoplasm Invasiveness; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology; D014701:Veratridine / Q000494:pharmacology
D062557:NAV1.6 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; C100681:SCN8A protein, human; C491046:Scn8a protein, mouse; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D013779:Tetrodotoxin; D014701:Veratridine; D002118:Calcium