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The 9 1 Integrin Enhances Cell Migration by Polyamine-Mediated

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The 9 1 Integrin Enhances Cell Migration by Polyamine-Mediated The ␣9␤1 integrin enhances cell migration by polyamine-mediated modulation of an inward-rectifier potassium channel Gregory W. deHart*, Taihao Jin†, Diane E. McCloskey‡, Anthony E. Pegg‡, and Dean Sheppard*§ *Lung Biology Center, Department of Medicine, and †Howard Hughes Medical Institute, Department of Physiology, University of California, San Francisco, CA 94143; and ‡Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033 Edited by Lily Y. Jan, University of California School of Medicine, San Francisco, CA, and approved March 11, 2008 (received for review August 27, 2007) The ␣9␤1 integrin accelerates cell migration through binding of dependent enhancement of cell migration (5). To systematically eval- spermidine/spermine acetyltransferase (SSAT) to the ␣9 cytoplasmic uate the role of SSAT catalytic activity, we studied six catalytically domain. We now show that SSAT enhances ␣9-mediated migration inactive mutants of SSAT (20, 21) with mutations scattered throughout specifically through catabolism of spermidine and/or spermine. Be- the protein. Each of these bound to the ␣9 cytoplasmic domain (Fig. cause spermine and spermidine are effective blockers of K؉ ion efflux 1a), but expression of any of three point mutants or three C-terminal -through inward-rectifier K؉ (Kir) channels, we examined the involve- truncation mutants caused comparable and significant dominant ment of Kir channels in this pathway. The Kir channel inhibitor, negative inhibition of migration on the ␣9-specific ligand, TNfn3RAA barium, or knockdown of a single subunit, Kir4.2, specifically inhib- (Fig. 1b) (5). A catalytically inactive mutant (K87A) did not affect ited ␣9-dependent cell migration. ␣9␤1 and Kir4.2 colocalized in focal migration of cells expressing a chimeric ␣ subunit containing the ␣9 adhesions at the leading edge of migrating cells and inhibition or extracellular and transmembrane domains and the ␣5 integrin cyto- knockdown of Kir4.2 caused reduced persistence and an increased plasmic domain (␣9␣5) (Fig. 1c). number of lamellipodial extensions in cells migrating on an ␣9␤1 ligand. These results identify a pathway through which the ␣9 ␣9-Dependent Migration Requires the Presence and Catabolism of integrin subunit stimulates cell migration by localized polyamine Polyamines. SSAT acetylates its polyamine substrates, spermine and catabolism and modulation of Kir channel function. spermidine, the rate-limiting step in polyamine catabolism (12, 13). To examine the specific role of polyamines in ␣9-mediated migra- ␣ he ␣9␤1 integrin is widely expressed and binds to a number of tion, we treated cells with -difluoromethylornithine (DFMO), an Tligands, including the extracellular matrix protein tenascin-C, inhibitor of the enzyme ornithine decarboxylase (ODC) [support- the vascular cell adhesion molecule, VCAM-1, and several mem- ing information (SI) Fig. S1a]. ODC catalyzes the conversion of the bers of the ADAM family (1–3). ␣9␤1 mediates enhanced cell polyamine precursor ornithine to the low order polyamine pu- migration, an effect that specifically depends on the ␣9 cytoplasmic trescine. Chinese hamster ovary (CHO) cells were treated for 2 days domain (4, 5). The closely related integrin ␣4 subunit also accel- with 5 mM DFMO, and polyamine levels were analyzed by high- erates cell migration, through reversible interaction with the scaf- performance liquid chromatography (22). As previously reported in folding protein, paxillin (6, 7). The mechanism by which the ␣9 ref. 23, DFMO substantially reduced both spermidine and pu- subunit cytoplasmic domain accelerates cell migration is completely trescine levels, whereas spermine levels were unaffected. Addition different (4, 5), and requires binding of the enzyme, spermidine/ of putrescine (100 ␮M) to DFMO-treated cells restored the levels spermine-N1-acetyltransferase (SSAT) (5). The mechanisms by of spermidine and putrescine (Fig. 2a). Migration of cells expressing which SSAT binding modulates migration were until now unknown. the wild-type ␣9 integrin subunit was significantly inhibited by SSAT specifically catalyzes catabolism of the higher order poly- DFMO, whereas migration of cells expressing the ␣9␣5 chimera amines, spermidine and spermine, to the lower order polyamine, was not affected (Fig. 2b). Putrescine rescued the DFMO-induced putrescine (8), thereby increasing intracellular levels of putrescine, decrease in migration (Fig. 2b), demonstrating the dependence of and decreasing those of spermidine and spermine (9, 10). ␣9 integrin-mediated migration on polyamines. Spermine and spermidine are potent blockers of outward po- Enhancement of Migration Is Not due to Increased Concentrations of tassium (Kϩ) currents from inward rectifier Kϩ (Kir) channels Acetylated Polyamine Intermediates. The acetylation of spermine (11–13). These channels conduct larger inward currents at mem- and spermidine by SSAT causes temporary increases in acetylated brane voltages below the resting potential than outward currents at polyamine intermediates. To determine whether these intermedi- more positive voltages. The long, positively charged polyamines, ates or the subsequent catabolism of higher order polyamines is spermine (ϩ4) or spermidine (ϩ3), mediate rectification by binding responsible for ␣9-mediated migration, we evaluated the effects of to negatively charged residues in the channel pore (12, 14). The catabolized polyamine putrescine (ϩ2) is less effective at blocking knockdown of polyamine oxidase (PAO), the enzyme responsible outward flow of Kϩ ions (11, 12). Although Kir channels have been for catabolism of acetylated polyamines. Two of three PAO siRNAs implicated in the regulation of many different physiological pro- cesses, including heart rate and membrane excitability (15), there Author contributions: G.W.d. and D.S. designed research; G.W.d. and T.J. performed are currently no reports suggesting their involvement in the regu- research; T.J., D.E.M., and A.E.P. contributed new reagents/analytic tools; G.W.d., T.J., lation of cell migration. However, numerous studies have suggested D.E.M., and A.E.P. analyzed data; and G.W.d. and D.S. wrote the paper. that Kϩ efflux is a critical factor in modulating cell migration The authors declare no conflict of interest. (16–19). In this study, we show that both the catalytic activity of This article is a PNAS Direct Submission. SSAT and the downstream catabolism of polyamines are specifi- Freely available online through the PNAS open access option. cally required for ␣9␤1-mediated migration, and identify a specific See Commentary on page 7109. role for the Kir channel subunit, Kir4.2, in mediating this effect. §To whom correspondence should be addressed at: Box 2922, University of California, San Francisco, CA 94143-2922. E-mail: [email protected]. Results This article contains supporting information online at www.pnas.org/cgi/content/full/ SSAT Catalytic Activity Is Crucial for ␣9-Dependent Migration. Binding 0708044105/DCSupplemental. of SSAT to the ␣9 integrin cytoplasmic domain is required for ␣9- © 2008 by The National Academy of Sciences of the USA 7188–7193 ͉ PNAS ͉ May 20, 2008 ͉ vol. 105 ͉ no. 20 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708044105 Downloaded by guest on September 30, 2021 a wt R101A/ R7A K87A R142 K161 L164 b c E152K Stop Stop Stop 160 * 125 SEE COMMENTARY 120 100 20 α9α9α9 GST ------------------------------------------------------------- 75 pull-down 80 15 ** 50 * 40 25 20 0 input of control) (% Migration 0 15 α9α9α9 of control) (% Migration control K87A control K87A CHO wt R101A/ R142 K161 L164 R7A K87A α9 α9α5 SSAT E152K Stop Stop Stop CHO CHO Fig. 1. Expression of catalytic mutants of SSAT inhibits ␣9-dependent migration. (a) 35S-labeled wild-type SSAT (wt), R101A/E152K, R7A, K87A point mutants, and truncation mutants containing C-terminal stop codons at R142 (R142Stop), K161 (K161Stop), or L164 (L164Stop) were produced by in vitro transcription, mixed with ␣9 cytoplasmic domain fused to GST and glutathione Sepharose 4B, and bound proteins were analyzed by SDS/PAGE and autoradiography. (b) CHO cells expressing Ϫ Ϫ ␣9 integrin subunit alone or with SSAT catalytic mutants migrated for 3 h across filters coated with 5 ␮g/ml TNfn3RAA. *, P ϭ 2 ϫ 10 9; **, P ϭ 1.5 ϫ 10 15.(c) Migration of CHO cells expressing ␣9or␣9␣5 integrin subunits alone, or with the SSAT catalytic point mutant K87A, analyzed after3hasinb. Data in b and c are expressed as mean Ϯ SD. *, P ϭ 0.001. significantly decreased mRNA levels in mouse embryonic fibroblasts migration in ␣9-expressing cells with knockdown of Kir4.2 (Fig. 4b). (MEFs) and each of these significantly decreased migration of cells In addition, treatment with putrescine either alone or for recon- expressing wild-type ␣9 (Fig. 2c). siRNA3, which was ineffective in stitution of polyamine levels in DFMO-treated cells did not mod- reducing PAO mRNA levels, had only a minor effect on migration (Fig. ulate migration of cells expressing shRNA to Kir4.2. Expression of 2c), and no siRNA affected the migration of cells expressing an ␣9␣4 wild-type Kir4.2 completely rescued migration, whereas equivalent chimera (Fig. 2c). Addition of putrescine to siRNA-treated cells did not expression of a mutant with impaired rectification (E157N) (Figs. rescue the effects of PAO knockdown (Fig. 2d). S2a and S4e) only partially restored migration (Fig. 4c). Expression of either wild-type or mutant proteins did not affect migration of Inward Rectifier Potassium Channel Function Is Important for ␣9 ␣9␣4-expressing cells (Fig. S4f). These data support a role for Integrin-Mediated Migration. One well characterized function of Kir4.2 channel rectification downstream of SSAT-mediated catab- spermine and spermidine is inward rectification of Kir channels (12). olism of polyamines. We therefore evaluated the expression levels of Kir channel subunits in To determine how Kir4.2 modulates cell migration, we per- CELL BIOLOGY ␣9-expressing MEFs. MEFs expressed at least eight Kir channel formed scratch wound assays. ␣9-expressing cells demonstrated subunits, including Kir 1.1, 2.1, 2.4, 3.2, 3.4, 4.2, 6.1, and 6.2 (Fig.
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