Pleiotrophin stimulates endothelial cell migration by affecting moesin phosphorylation and interaction with alpha v beta 3 integrin

E. Papadimitriou1, S. Tsirmoula1, M. Koutsioumpa1, L. Skondra1, S. Skandalis2, C. Mikelis1. 1Dept of Pharmacy, University of Patras, Patras, GREECE, 2Dept of Chemistry, University of Patras, Patras, GREECE.

Introduction: Pleiotrophin (PTN) is a heparin-binding growth factor that through its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and alpha v beta 3 (ανβ3) integrin regulates endothelial cell migration1,2. Although interaction between RPTPβ/ζ and ανβ3 is a prerequisite for the stimulatory effect of PTN through s-Src, PI3K and cell surface nucleolin on endothelial cell migration2,3, the complete signaling pathway activated by PTN still needs to be elucidated. Moesin is a member of the ERM protein family that appears to function as a cross-linker between cell membrane and actin-based cytoskeleton and to be important for cell-cell recognition and cell movement4. By performing MALDI-TOF analysis in endothelial cells’ immunoprecipitates against ανβ3 and PTN, moesin has been identified as an interacting protein in both cases, but not in immunoprecipitates against RPTPβ/ζ. In the present work, we verified this finding and studied its functional significance.

Methods: Human umbilical vein endothelial cells (HUVEC) and Chinese hamster ovary (CHO) cells expressing wild type β3 (wtβ3CHO) or mutant β3 where Tyr773 has been substituted to Phe (mutβ3CHO) were used. The interaction between moesin and ανβ3 or PTN was verified by IP/Western blot and proximity ligation assays. Moesin localization was studied by immunofluorescence. Down-regulation of moesin was performed by siRNA. Migration was estimated by a modified Boyden chamber assay. Pharmacological inhibitors were used to study involvement of signaling molecules.

Results: Moesin localizes mainly on the surface of endothelial cells, where it interacts with ανβ3. Upon stimulation with PTN, moesin phosphorylation is increased through RPTPβ/ζ, c-Src and PI3K, but its cell membrane localization, as well as its interaction with ανβ3 diminishes. Down-regulation of moesin by siRNA in endothelial cells leads to increased cell migration and in that case, PTN cannot cause further increase. This is in line with the increased cell migration of mutβ3CHO compared with wtβ3CHO cells, in which decreased moesin-ανβ3 interaction is also observed and PTN cannot further increase cell migration.

Conclusion: Interaction of moesin with β3Tyr773 has a negative role in endothelial cell migration, which is neutralized when either moesin or β3Tyr773 are phosphorylated through RPTPβ/ζ, c-Src and PI3K activation upon PTN stimulation. Involvement of cyclin-dependent kinase 5 and Rac1 will be also discussed.

References: 1. Polykratis A et al. (2005). J Biol Chem 280: 22454-22461. 2. Mikelis C et al. (2009). FASEB J 23:459-469. 3. Koutsioumpa M et al. (2013). J Biol Chem 288:43-54.4. Neisch AL et al. (2011). Curr Opin Cell Biol 23: 377-382.