RhoA/ROCK and Cdc42 regulate cell-cell contact and N-cadherin protein level during neurodetermination of P19 embryonal stem cells.
RhoGTPases regulate actin-based signaling cascades and cellular contacts. In neurogenesis, their action modulates cell migration, neuritogenesis, and synaptogenesis. Murine P19 embryonal stem cells differentiate to neurons upon aggregation in the presence of retinoic acid, and we previously showed that RhoA and Cdc42 RhoGTPases are sequentially up-regulated during neuroinduction, suggesting a role at this very early developmental stage. In this work, incubation of differentiating P19 cells with C3 toxin resulted in decreased aggregate cohesion and cadherin protein level. In contrast, C3 effects were not observed in cells overexpressing recombinant dominant active RhoA. On the other hand, C3 did not affect cadherin in uninduced cells and their postmitotic neuronal derivatives, respectively expressing E- and N-cadherin. RhoA is thus influential on cell aggregation and cadherin expression during a sensitive time window that corresponds to the switch of E- to N-cadherin. Cell treatment with Y27632 inhibitor of Rho-associated-kinase ROCK, or advanced overexpression of Cdc42 by gene transfer of a constitutively active form of the protein reproduced C3 effects. RhoA-antisense RNA also reduced cadherin level and the size of cell aggregates, and increased the generation of fibroblast-like cells relative to neurons following neuroinduction. Colchicin, a microtubule disrupter, but not cytochalasin B actin poison, importantly decreased cadherin in neurodifferentiating cells. Overall, our results indicate that the RhoA/ROCK pathway regulates cadherin protein level and cell-cell interactions during neurodetermination, with an impact on the efficiency of the process. The effect on cadherin seems to involve microtubules. The importance of correct timing of RhoA and Cdc42 functional expression in neurogenesis is also raised.
Laboratory of Developmental Neuroendocrinology, Département de chimie et biochimie, Université du Québec à Montréal, C.P. 8888, Succ. Centre-ville, Montreal, Quebec, H3C 3P8, Canada.
Isabel
I
Laplante
Richard
R
Béliveau
Joanne
J
Paquin
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D015820:Cadherins / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002449:Cell Aggregation / Q000187:drug effects / Q000502:physiology; D002450:Cell Communication / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D003176:Complement C3 / Q000494:pharmacology; D016373:DNA, Antisense / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004622:Embryo, Mammalian; D005786:Gene Expression Regulation / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013234:Stem Cells / Q000502:physiology*; D014162:Transfection / Q000379:methods; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
D015820:Cadherins; D003176:Complement C3; D016373:DNA, Antisense; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein