Abstract
Purpose: :
Numerous studies have demonstrated that Pinin (Pnn), functions as a critical mediator of various multiprotein complexes involved in regulation of gene expression, pre-mRNA splicing, and cell adhesion. Our recent studies suggest that Pnn is involved in the regulation of Wnt signaling pathway and NCC migration. We now explore the involvement of Pnn in the Wnt Pathway.
Methods: :
Pnn hypomorphic and conditional knock-out mice were utilized to explore for perturbations in expression of Wnt-related and downstream Wnt genes. HCET cells modified to stably express exogenous Pnn with two tandem epitope tags (HA and Flag) were utilized to immuno-isolate Pnn nuclear complexes.
Results: :
Consistent with our previous reports, eyes of both hypomorphic and conditional knock out of Pnn exhibited enhanced β-catenin expression. In addition a number of downstream Wnt genes also showed elevated expression, such as Axin2. Immuno-isolation of nuclear complexes from HCET revealed that Pnn was indeed in complex with a number of splicing and transcription related proteins such as; SR proteins, CtBP, SAP18, and RNA-helicase p68. Most interestingly, however, further examination of these complexes revealed the presence of β-catenin and GSK3. These data were striking because previous isolation and analyses of Pnn complexes from Hela cells did not reveal β-catenin. In addition inhibition of GSK3 activity with LiCl or inhibition of proteosome with MG-132 resulted in enhanced Pnn-p68 and Pnn-CtBP2 association.
Conclusions: :
These data lend strong support to the contention that Pnn is directly involved in the Wnt pathway, through its association with β-catenin. The results also suggest that the fundamental roles that Pnn plays in the establishing and maintenance of epithelia may be tied to these molecular interactions.
Keywords: cornea: basic science • development • gene/expression