Degrading Polyamines for Migration

Abstract

Integrins are heterodimeric transmembrane proteins that mediate cell adhesion and migration through various mechanisms. Binding of the cytoplasmic domain of the integrin α9 subunit to the spermidine/spermine- N 1 -acetyltransferase (SSAT)--which induces the breakdown of the higher-order polyamines spermidine and spermine to the lower-order polyamine putrescine--is required for migration mediated by the α9β1 integrin. deHart et al. (see commentary by Vandenberg) report that α9 recruitment of SSAT to focal adhesions affects cell migration by modulating the activity of an inward rectifier K + (Kir) channel. Both wild-type and catalytically inactive forms of in vitro-translated SSAT bound to tagged α9 in a pull-down assay. Expression of any of the catalytically inactive forms in α9-expressing Chinese hamster ovary (CHO) cells prevented migration on an α9-specific ligand in a dominant-negative fashion. This effect was specific to α9, because migration mediated by a chimeric α9α5 protein, in which the intracellular domain of α9 was replaced with the intracellular domain of α5, was not affected by expression of the catalytically inactive SSATs. Depleting intracellular spermidine concentrations, using a combination of the ornithine decarboxylase inhibitor DFMO and putrescine, inhibited migration of CHO cells that overexpressed wild-type α9 but not those expressing the α9α5 chimera. Unexpectedly, restoring spermidine concentration by addition of putrescine in DFMO-treated cells restored migration. Furthermore, in mouse embryo fibroblasts (MEFs) overexpressing α9, but not those overexpressing the chimeric α9α5, migration was reduced when the expression of polyamine oxidase (PAO), an enzyme required for polyamine metabolism downstream of SSAT, was knocked down by small interfering RNAs (siRNAs). Adding putrescine did not restore migration to the PAO-deficient cells. Thus, the results from experiments in which global intracellular polyamine concentrations were altered with pharmacological agents were complex but may indicate that local changes in cellular polyamine concentrations at the focal adhesion regulate migration. K + efflux is implicated in cell migration, and Kir channels are inhibited by spermidine and spermine; thus, SSAT may modulate polyamine concentrations to stimulate migration. Indeed, treating α9- (but not α9α4- or α9α5-) expressing MEFs with Ba 2+ to inhibit Kir channels or with a α9β1-blocking antibody decreased migration of cells on an α9-specific ligand but had no effect on adhesion of cells to nonintegrin-specific substrates. Of the eight Kir subunits detected in MEF cells, only Kir4.2 appeared to have a role in α9-mediated migration. Knockdown of Kir4.2 by a short hairpin RNA (shRNA) or inhibition of Kir channels with Ba 2+ in either α9-expressing MEFs or human microvascular endothelial cells, which natively express Kir4.2 and α9β1, impaired migration on α9 substrates. In the MEFs expressing α9, the effects of DFMO and Kir4.2 shRNA treatment were not additive, suggesting that polyamines regulate cell migration through Kir4.2. In an in vitro wound assay, α9-expressing MEF cells treated with Ba 2+ , Kir4.2 shRNA, or both migrated shorter distances than untreated cells and extended multiple leading-edge cytoplasmic projections, whereas untreated cells usually extended only one lamellopodium. Fluorescently tagged α9 and Kir4.2 colocalized to structures that looked like focal adhesions, but only at the leading edge of MEFs. These results suggest that the α9 subunit of integrins recruits SSAT to focal adhesions, where it decreases the local concentration of higher-order polyamines; this may relieve repression of Kir channels, thus initiating an outward K + flow. The authors model that the K + efflux may cause the influx of Ca 2+ , which is known to stimulate migration in other contexts. G. W. deHart, T. Jin, D. E. McCloskey, A. E. Pegg, D. Sheppard, The α9β1 integrin enhances cell migration by polyamine-mediated modulation of an inward-rectifier potassium channel. Proc. Natl. Acad. Sci. U.S.A. 105 , 7188-7193 (2008). [Abstract] [Full Text] C. A. Vandenberg, Integrins step up the pace of cell migration through polyamines and potassium channels. Proc. Natl. Acad. Sci. U.S.A. 105 , 7109-7110 (2008). [Full Text]