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Wnt5a signals through the noncanonical Wnt pathway in thymocytes. Western blot on Wnt5a-infected or empty vector–infected (e.v.) NFC cell protein extracts (A) and on protein extracts of Wnt5a+/+ or Wnt5a−/− E18.5 thymocytes (D) with primary antibodies against phosphorylated PKC, β-catenin, or α-tubulin. (B) CamKII activity assays performed on Wnt5a-infected or e.v. NFC cells. (C) Representative Western blot displaying inhibition of activated PKC and β-catenin levels in fetal thymocytes. (D) CamKII assays performed on E18 fetal thymocytes reveals reduced CamKII activity in Wnt5a−/− cells. All experiments in A–D were performed three times with similar results. Error bars represent the SD. P < 0.05. (E) Addition of Wnt5a inhibits β-catenin activation of TOPFLASH reporter in NFC cells. RLU, relative luciferase activity. Results are the mean of three independent transfections. This assay was repeated twice, with similar qualitative results. Error bars represent the SD. P < 0.05.
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fig5: Wnt5a signals through the noncanonical Wnt pathway in thymocytes. Western blot on Wnt5a-infected or empty vector–infected (e.v.) NFC cell protein extracts (A) and on protein extracts of Wnt5a+/+ or Wnt5a−/− E18.5 thymocytes (D) with primary antibodies against phosphorylated PKC, β-catenin, or α-tubulin. (B) CamKII activity assays performed on Wnt5a-infected or e.v. NFC cells. (C) Representative Western blot displaying inhibition of activated PKC and β-catenin levels in fetal thymocytes. (D) CamKII assays performed on E18 fetal thymocytes reveals reduced CamKII activity in Wnt5a−/− cells. All experiments in A–D were performed three times with similar results. Error bars represent the SD. P < 0.05. (E) Addition of Wnt5a inhibits β-catenin activation of TOPFLASH reporter in NFC cells. RLU, relative luciferase activity. Results are the mean of three independent transfections. This assay was repeated twice, with similar qualitative results. Error bars represent the SD. P < 0.05.

Mentions: To examine if Wnt5a signals via the noncanonical or canonical Wnt pathway in thymocytes, Wnt–Ca2+ signaling and β-catenin stabilization were analyzed in Wnt5a-transduced NFC cells. The levels of activated PKC (Fig. 5 A) and CamKII activity (Fig. 5 B) were clearly elevated in Wnt5a-expressing cells. Furthermore, β-catenin levels were not stimulated, but rather were consistently reduced by Wnt5a expression (Fig. 5 A). These data indicate that Wnt5a is signaling through the noncanonical pathway in thymocytes. To confirm these results in vivo, total protein was extracted from E17.5 thymocytes isolated from WT and Wnt5a−/− littermates, and relative amounts of proteins in the Wnt signaling pathway were assayed. Consistent with the in vitro results, the amount of activated PKC was clearly reduced in Wnt5a−/− thymocytes relative to controls (Fig. 5 C). In addition, β-catenin levels were significantly elevated in Wnt5a−/− thymocytes. In addition, CamKII activity was decreased in thymocytes isolated from thymus of Wnt5a−/− embryos (E18). These data indicate that Wnt5a activates the Wnt–Ca2+ pathway in developing thymocytes in vivo, and may inhibit canonical Wnt signaling. Because β-catenin is normally expressed at the highest level in DN thymocytes and Wnt5a−/− mice show a marginal reduction in DN thymocytes, the observed increase in β-catenin amounts cannot be attributed to altered thymic subset composition in Wnt5a−/− thymus. As β-catenin/TCF1 signaling has been shown to ensure the survival of DP thymocytes (27), Wnt5a may normally contribute to the regulation of cell survival by countering the antiapoptotic function of β-catenin/TCF1. However, the consequence of this antagonism does not appear to involve the TCF1 target Bcl-XL, as Bcl-XL levels appear unchanged when Wnt5a is not present (Fig. 3 B). Instead, Wnt5a may promote apoptosis by up-regulating Bax gene expression. To determine if Wnt5a can induce alterations in β-catenin activity, NFC cells that were mock transfected or transfected with a Wnt5a expression vector were cotransfected with a β-catenin expression vector and TOPFLASH, which is a reporter plasmid–bearing luciferase gene under transcriptional control of a β-catenin/TCF1–responsive promoter. A plasmid encoding Renilla luciferase was also cotransfected as a control for transfection efficiency. Expression of the TOPLASH reporter was not detectible in the absence of cotransfected β-catenin in these cells. Thus, the β-catenin/TCF1–responsive promoter is exclusively dependent on exogenous β-catenin activity. Expression of Wnt5a significantly inhibited activation of the promoter after cotransfection with β-catenin, confirming that Wnt5a inhibits the activity of β-catenin/TCF1 in T cells (Fig. 5 E),

Noncanonical Wnt signaling promotes apoptosis in thymocyte development

Liang H, Coles AH, Zhu Z, Zayas J, Jurecic R, Kang J, Jones SN - J. Exp. Med. (2007)

Bottom Line: Our findings reveal that Wnt5a produced in the thymic stromal epithelium does not alter the development of progenitor thymocytes, but regulates the survival of alphabeta lineage thymocytes.Conversely, Wnt5a deficiency results in the inhibition of PKC activation, decreased CamKII activity, and elevation of beta-catenin amounts in thymocytes.These results indicate that Wnt5a induction of the noncanonical Wnt-Ca(2+) pathway alters canonical Wnt signaling and is critical for normal T cell development.

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01545, USA.

ABSTRACT
The Wnt-beta-catenin signaling pathway has been shown to govern T cell development by regulating the growth and survival of progenitor T cells and immature thymocytes. We explore the role of noncanonical, Wnt-Ca(2+) signaling in fetal T cell development by analyzing mice deficient for Wnt5a. Our findings reveal that Wnt5a produced in the thymic stromal epithelium does not alter the development of progenitor thymocytes, but regulates the survival of alphabeta lineage thymocytes. Loss of Wnt5a down-regulates Bax expression, promotes Bcl-2 expression, and inhibits apoptosis of CD4(+)CD8(+) thymocytes, whereas exogenous Wnt5a increases apoptosis of fetal thymocytes in culture. Furthermore, Wnt5a overexpression increases apoptosis in T cells in vitro and increases protein kinase C (PKC) and calmodulin-dependent kinase II (CamKII) activity while inhibiting beta-catenin expression and activity. Conversely, Wnt5a deficiency results in the inhibition of PKC activation, decreased CamKII activity, and elevation of beta-catenin amounts in thymocytes. These results indicate that Wnt5a induction of the noncanonical Wnt-Ca(2+) pathway alters canonical Wnt signaling and is critical for normal T cell development.

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