ABSTRACT
The sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), can induce or inhibit cellular migration. The intermediate filament protein vimentin is an inducer of migration and a marker for epithelial–mesenchymal transition. Given that keratin intermediate filaments are regulated by SPC, with consequences for cell motility, we wanted to determine whether vimentin is also regulated by sphingolipid signalling and whether it is a determinant for sphingolipid-mediated functions. In cancer cells where S1P and SPC inhibited migration, we observed that S1P and SPC induced phosphorylation of vimentin on S71, leading to a corresponding reorganization of vimentin filaments. These effects were sphingolipid-signalling-dependent, because inhibition of either the S1P2 receptor (also known as S1PR2) or its downstream effector Rho-associated kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) nullified the sphingolipid-induced effects on vimentin organization and S71 phosphorylation. Furthermore, the anti-migratory effect of S1P and SPC could be prevented by expressing S71-phosphorylation-deficient vimentin. In addition, we demonstrated, by using wild-type and vimentin-knockout mouse embryonic fibroblasts, that the sphingolipid-mediated inhibition of migration is dependent on vimentin. These results imply that this newly discovered sphingolipid–vimentin signalling axis exerts brake-and-throttle functions in the regulation of cell migration.
Footnotes
↵* These authors contributed equally to this work
Competing interests
The authors declare no competing or financial interests.
Author contributions
C.L.H. and K.K. devised the key concepts of the experimental plan under the guidance of J.E.E. and K.T. The Boyden chamber migrations, all western blotting and blot analysis and qPCR experiments were performed by K.K. Live-cell migration imaging, tracking and analysis, confocal imaging and analysis were done by C.L.H. with help from K.K. The mass spectrometry was performed and analysed by S.Y.I. The vimentin phospho-antibodies were generated by H.G. and M.I., who also provided manuscript advice. K.I. performed the cell perimeter and FRAP experiments and analysis. E.F. assisted with confocal imaging and analysis. C.L.H., K.K., S.Y.I., K.I., E.F. generated the figures. C.L.H. and K.K. co-wrote the article with J.E.E. and K.T. The J.E.E. and K.T. laboratories had equal contribution.
Funding
This study was supported by the Sigrid Jusélius Foundation; the Liv och Hälsa Foundation; The Academy of Finland; the Centre of Excellence in Cell Stress and Molecular Ageing (Åbo Akademi University); Åbo Akademi University; Finnish Cultural Foundation; Ida Montin Foundation; the K. Albin Johansson Foundation; the Tor, Joe and Pentti Borg Foundation; the Arvid and Greta Olin Foundation; and the Receptor Research Program (Åbo Akademi University and the University of Turku).
Supplementary material
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.160341/-/DC1
- Received July 23, 2014.
- Accepted April 20, 2015.
- © 2015. Published by The Company of Biologists Ltd
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