Regulation of Podosome Formation in Macrophages by a Splice Variant of the Sodium Channel SCN8A*
- Michael D. Carrithers‡§1,
- Gouri Chatterjee‡,
- Lisette M. Carrithers‡2,
- Roosevelt Offoha‡,
- Uzoma Iheagwara‡,
- Christoph Rahner¶,
- Morven Graham¶ and
- Stephen G. Waxman‡∥
- Departments of ‡Neurology and ¶Cell Biology and §Program in Human and Translational Immunology, Yale University School of Medicine, New Haven, Connecticut 06520-8018 and the ∥Center for Neuroscience and Neuroregeneration Research, Veterans Administration Connecticut Healthcare, West Haven, Connecticut 06518
- 1 To whom correspondence should be addressed: Dept. of Neurology, University of Wisconsin School of Medicine and Public Health, 600 N. Highland Ave., Madison, WI 53792. Tel.: 608-265-0329; Fax: 608-263-0412; E-mail: carrithers{at}neurology.wisc.edu.
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.
Footnotes
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↵3 The abbreviations used are: TTX, tetrodotoxin; m-CSF, macrophage colony-stimulating factor; MCP-1, monocyte chemotractant protein; NMDG, N-methyl-d-glucamine; SBFI, sodium binding benzofuran isopthalate; shRNA, small hairpin RNA; FBS, fetal bovine serum; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution; RFU, relative fluorescent unit.
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The nucleotide sequence(s) reported in this paper has been submitted to the Gen-Bank™/EBI Data Bank with accession number(s) FJ11941.
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↵* This work was supported, in whole or in part, by the National Institutes of Health. This work was also supported by the National Multiple Sclerosis Society, the Nancy Davis Foundation, and a Dana Foundation award in Clinical Hypotheses in Neuroimmunology (to M. D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2.
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↵2 Current address: Dept. of Neurology, University of Wisconsin School of Medicine and Public Health, 600 N. Highland Ave., Madison, WI 53792.
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- Received March 7, 2008.
- Revision received January 7, 2009.
- The American Society for Biochemistry and Molecular Biology, Inc.
