Abstract 3945: Atrophin Proteins Interact With The Fat1 Cadherin And Regulate Migration And Orientation In Vascular Smooth Muscle Cells
The mammalian Fat1 cadherin regulates vascular smooth muscle cell (VSMC) growth and migration, but the molecular mechanisms underlying these effects are not well understood. In Drosophila, the related protein Fat interacts genetically and physically with Atrophin to regulate planar cell polarity. We hypothesized that a similar interaction between Fat1 and one or more of the 3 mammalian Atrophin isoforms (Atr1, Atr2S, and Atr2L) might contribute to control of VSMC activities important in vascular remodeling. Co-immunoprecipitation studies demonstrated interaction between endogenous Atr and Fat1 in VSMC lysates. Mapping studies with co-transfected epitope-tagged Atr1 or Atr2L and Fat1 derivatives indicated that interaction required Fat1 aa residues 4300 – 4400 (overlapping with a Fat1-β-catenin interaction domain we characterized previously), as well as an intact Atrophin-box in the Atrophin C-terminal domains. Knockdown of Atrs by siRNA yielded no changes in VSMC growth, but complex effects on VSMC migration, which was impaired by Atr1 knockdown and enhanced by Atr2L knockdown. Importantly, enhanced migration with Atr2L knockdown was Fat1-dependent. In assays of cellular orientation after scratch wounding of confluent monolayers, we found that Atr1 knockdown impaired orientation of cells along the wound margin toward the denuded area (37.8% positively-oriented microtubule organizing center (MTOC), vs. 47.8% in control, P<.05), while Atr2L knockdown enhanced it (53.6% positively-oriented MTOC vs. 39.7% in control, P<.05). Together with the ~4 –5-fold induction of Atr1 and Atr2S expression after arterial injury and by pro-migratory factors, these findings suggest a model in which Atr1 and Atr2S, together with Fat1, act to enhance VSMC migration and orientation by opposing the function of Atr2L. Because Atr2L, but not Atr1 or Atr2S, bears multiple motifs associated with chromatin remodeling and gene regulation, these effects on migration and orientation may reflect changes in VSMC gene expression that promote a migratory phenotype. These findings demonstrate physical and functional interaction of Fat1 intracellular domain with Atrophins, and point to a novel signaling mechanism underlying Fat1 effects on VSMC orientation and migration.