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PNAS 105 (20): 7188-7193

Copyright © 2008 by the National Academy of Sciences.

The {alpha}9β1 integrin enhances cell migration by polyamine-mediated modulation of an inward-rectifier potassium channel

Gregory W. deHart*, Taihao Jin{dagger}, Diane E. McCloskey{ddagger}, Anthony E. Pegg{ddagger}, and Dean Sheppard*,§

*Lung Biology Center, Department of Medicine, and {dagger}Howard Hughes Medical Institute, Department of Physiology, University of California, San Francisco, CA 94143; and {ddagger}Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033


Figure 1
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  		Fig. 1. Expression of catalytic mutants of SSAT inhibits {alpha}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 {alpha}9 cytoplasmic domain fused to GST and glutathione Sepharose 4B, and bound proteins were analyzed by SDS/PAGE and autoradiography. (b) CHO cells expressing {alpha}9 integrin subunit alone or with SSAT catalytic mutants migrated for 3 h across filters coated with 5 µg/ml TNfn3RAA. *, P = 2 x 10–9; **, P = 1.5 x 10–15. (c) Migration of CHO cells expressing {alpha}9 or {alpha}9{alpha}5 integrin subunits alone, or with the SSAT catalytic point mutant K87A, analyzed after 3 h as in b. Data in b and c are expressed as mean ± SD. *, P = 0.001.

 

Figure 2
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  		Fig. 2. Modulation of polyamine levels affects {alpha}9-dependent enhanced migration. (a) Levels of polyamines from cells treated for 2 d with 5 mM DFMO with or without 100 µM putrescine. (b) {alpha}9- or {alpha}9{alpha}5-expressing CHO cells were treated as in a, and migration on TNfn3RAA was measured after 3 h. *, P = 2.2 x 10–10; **, P = 9.8 x 10–8. (c) {alpha}9- or {alpha}9{alpha}4-expressing MEFs were treated or not with three different siRNAs to PAO and induced to migrate on TNfn3RAA. *, P = 2.0 x 10–4; **, P = 0.014. (d) Migration of {alpha}9-transfected MEF cells treated with siRNA to PAO as in c, with or without additional treatment with 100 µM putrescine. *, P = 8 x 10–16; **, P = 1.1 x 10–16.

 

Figure 3
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  		Fig. 3. Kir channel involvement in {alpha}9-dependent migration. (a) mRNA from MEF{alpha}9 cells was analyzed by RT-PCR by using primers to the indicated Kir subunits. (b) {alpha}9- and {alpha}9{alpha}4-expressing MEFs were induced to migrate on TNfn3RAA in the presence or absence of a range of Ba2+ concentrations, or the anti-{alpha}9β1 mAb, Y9A2. *, P = 1.06 x 10–5; **, P = 7.6 x 10–12; ***, P = 1.25 x 10–14. (c) MEF{alpha}9 cells were pretreated or not with 50 µg/ml Y9A2, 32 µM Ba2+, 100 nM TPNQ, 1 mM Glyb, or both TPNQ and Glyb and then induced to migrate on either 1% BSA (untreated cells) or TNfn3RAA. *, P = 1.4 x 10–15; **, P = 1.8 x 10–15.

 

Figure 4
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  		Fig. 4. Effect of knockdown of Kir4.2 on {alpha}9-dependent enhanced migration. (a) MEF{alpha}9 cells expressing pSicoR or shRNA to Kir4.2 were treated overnight or not with cre recombinase for shRNA excision before addition to TNfn3RAA-coated Transwell filters. Some control cells were treated with 32 µM Ba2+. Migration after 3 h is expressed as a percentage of migration of untreated cells. *, P = 0.0007; **, P = 0.02. (b) Control MEF{alpha}9 cells or cells expressing either pSicoR or Kir4.2 shRNA were treated for 2 d with 5 mM DFMO, 100 µM putrescine, or both and induced to migrate across Transwell filters coated with Tnfn3RAA. *, P = 0.04; **, P = 0.07. (c) Migration on Tnfn3RAA of wild-type or E157N mutant Kir4.2-expressing MEF{alpha}9 cells treated or not with Kir4.2 shRNA. *, P = 2.0 x 10–17; **, P = 9.1 x 10–6.

 

Figure 5
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  		Fig. 5. Morphological analysis of {alpha}9-dependent scratch wound migration. (a) Confluent MEF{alpha}9 cells or cells expressing shRNA to Kir4.2 were wounded with a pipette tip in the presence or absence of 32 µM Ba2+, and then imaged every 10 min for 15 h. Representative tracks of cell centroids (black dots) migrating into the wound space are shown. (b) Mean instantaneous velocity of the cells treated in a. *, P = 2.0 x 10–5. (c) Mean change in the angle of migration relative to the initial direction, as analyzed in b. *, P = 0.0003; **, P = 0.004; ***, P = 0.004. (d) Mean persistence in cell migration of the same cells analyzed in b and c. *, P = 0.0001; **, P = 0.007; ***, P = 0.009. (e) Images of individual cells and lamellipodia at the wound edge (arrows). (f) Quantification of the mean number of lamellipodial extensions >2 µm in width in cells treated as in a–d and imaged every 2 min for 2 h. *, P = 0.0008; **, P = 0.0008; ***, P = 0.0004.

 

Figure 6
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  		Fig. 6. Involvement of Kir4.2 subunits in {alpha}9-dependent cell migration of HMVECs. (a) RT-PCR of Kir channel subunits expressed in HMVECs. (b) HMVECs were treated with the {alpha}9β1 integrin-blocking antibody Y9A2 or with 32 µM Ba2+, and induced to migrate across filters coated with 5 µg/ml TNfn3RAA or FN, or 3 µg/ml VEGF-C. *, P = 0.01; **, P = 0.03; {dagger}, P = 0.14; #, P = 8.2 x 10–8; ##, P = 0.0005. (c) Uninfected HMVECs or cells expressing either pSicoR or Kir4.2 shRNA alone or also expressing Cre were induced to migrate across filters coated with 5 µg/ml TNfn3RAA. *, P = 0.30; **, P = 2.6 x 10–11.

 

Figure 7
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  		Fig. 7. The {alpha}9 integrin and Kir4.2 codistribute in focal adhesions at the leading edge of migrating cells. (a and d) {alpha}9 integrin-GFP localization in MEF cells at the edge of a scratch wound. (b and e) Kir4.2-mCherry distribution in the same cells as in a and d. (c) Overlay of {alpha}9-GFP and Kir4.2-mCherry images in a and b. (f) Overlay of {alpha}9-GFP and Kir4.2-mCherry images in d and e. Arrows, Colocalization of {alpha}9-GFP and Kir4.2-mCherry. Arrowheads, {alpha}9 integrin localization alone. (Scale bar, 10 µm.)

 

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