Wnt proteins are crucial for development/homeostasis by controlling cell fate including apoptosis (Moon RT et al. 1997). Three humanWnt13 isoforms were identified: the secreted Wnt13A, mitochondrial Wnt13B, and nuclear Wnt13C forms; and nuclear Wnt13 had an increased sensitivity to LPS/TNF-induced apoptosis in primary endothelial cells (EC); both Wnt13B and C mRNA contain two start codons (AUG+1 and +74), but the same protein encoded from AUG+74 by Wnt13C was expressed lower than Wnt13B (Struewing IT et al.2006). We hypothesize that during EC apoptosis, the nuclear Wnt13C expression is regulated translationally; nuclear Wnt13 favors apoptosis through regulating the activity/expression of apoptosis-related factors; Wnt13 isoforms may have differential effects on EC apoptosis and apoptosis-related factors.
1. The protein levels, but not the mRNA levels of Wnt13C were induced by apoptosis-inducers. And the Myc-tag insertion at the AUG+1 in Wnt13C mRNA inhibited its expression, indicating the RNA sequences/structures are critical. Therefore, nuclear Wnt13C is regulated during apoptosis at translational levels.
2. Nuclear Wnt13 increased caspase-3/7 expression with/without LPS, followed by an increase in LPS-induced caspase-3/7 cleavage; and nuclear Wnt13 upregulated the pro-apoptotic Bcl-2 family member Bim expression, suggesting that nuclear Wnt13 increased caspase activation through upregulating caspase and Bim expression. Wnt13 isoforms increased EC apoptosis with different strengths: nuclear > mitochondrial > secreted forms.
3. Both caspase-3 and Bim are FOXO target genes; and nuclear Wnt13 increased the nuclear localization of FOXOs, suggesting increased FOXO activity. Nuclear Wnt13 also upregulated SOD2, another FOXO target gene related to oxidative stress-resistance.
Nuclear Wnt13 did not increase FOXO activity at the SOD2 promoter, but increased the SOD2-intron 2 element luciferase activity upon LPS, where a novel putative FOXO site was found, implying intron 2 may be responsible for enhanced SOD2 transcription by nuclear Wnt13.
Altogether, our results pinpoint the interplay between the expression and functions of Wnt13 forms during EC apoptosis, forming a positive cycle further facilitating the apoptotic program completion, which is important for EC homeostasis.
| Identifer | oai:union.ndltd.org:uky.edu/oai:uknowledge.uky.edu:gradschool_diss-1756 |
| Date | 01 January 2009 |
| Creators | Tang, Tao |
| Publisher | UKnowledge |
| Source Sets | University of Kentucky |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of Kentucky Doctoral Dissertations |
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