Abstract
Wnt proteins play a key role in cell survival, cell proliferation, and cell fate during development. In endothelial cells, we identified the expression of Wnt13A, Wnt13B, and Wnt13C mRNAs, which are generated by alternative promoters and alternative RNA splicing. Wnt13A and Wnt13B proteins differ only in their N-terminal sequences. Wnt13A, a typical Wnt, is N-glycosylated and localized in the endoplasmic reticulum, with only a small fraction being secreted. Wnt13B proteins appear as a protein doublet, L-Wnt13B and S-Wnt13B, which are neither N-glycosylated nor secreted. Wnt13B proteins localized mainly to mitochondria, as demonstrated using detection in mitochondria enriched fractions and colocalization with Mitotracker and HSP60. A nuclear localization was also observed in 20% of Wnt13B-expressing cells. Both the N-terminal hydrophobic stretch (residues 1-17) and alpha-helix (residues 26-50) were the main determinants for Wnt13B mitochondrial targeting. Serial deletions of Wnt13B N-terminal sequences abolished its association with mitochondria and favored instead a nuclear localization. The production of S-Wnt13B was independent of the mitochondrial targeting but dependent on an alternative translation start corresponding to Met(74) in L-Wnt13B. The same translation start is used in Wnt13C mRNA to encode a protein undistinguishable from S-Wnt13B. S-Wnt13B when expressed alone localized to the nucleus like Wnt13C, whereas L-Wnt13B localized to mitochondria. Wnt13 nuclear forms increased the beta-catenin/T-cell factor activity in HEK293 cells and increased apoptosis in bovine aortic endothelial cells. Altogether our results demonstrate that, in addition to alternative promoters and RNA splicing, an alternative translation start in Wnt13B and Wnt13C mRNAs increases the complexity of both human wnt13 expression and functions.
Highlights
In mammals, 19 different Wnt family members displaying a conserved pattern of 21–23 cysteine residues have been identified so far
We have identified the expression of three different mRNA forms of the wnt13 gene in endothelial cells of different origins: the previously described Wnt13A/Wnt2B2 and Wnt13B/Wnt2B1 mRNA forms [20] and an additional Wnt13C mRNA generated by an alternative splicing that skips exon 2 (Fig. 1)
Two cDNAs corresponded to the previously described Wnt13A/Wnt2b2 mRNA transcribed from the P2 promoter and composed of exons 3–7 and Wnt13B/Wnt2b1 mRNA transcribed from the P1 promoter and composed of exons 1 and 2 and exons 4 –7 (Fig. 1, A–C)
Summary
19 different Wnt family members displaying a conserved pattern of 21–23 cysteine residues have been identified so far. During chick development of the retina, wnt was expressed in particular at the marginal tip of the retina, where it maintains undifferentiated progenitor cells in the ciliary marginal zone [21, 22] These effects of Wnt were associated with the activation of the canonical -catenin/LEF/ TCF signaling pathway [23]. Whereas Wnt13A is a classical Wnt protein, N-glycosylated and secreted, Wnt13B and Wnt13C are intracellular forms targeted to mitochondria and to the nucleus respectively Such different subcellular localizations for Wnt13A and Wnt13B proteins explain the differential activities previously observed for these two forms [25]. These results strengthen the importance of the processing of Wnt proteins in relationship with their targeting and intracellular activities
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