Hedgepeth CM et al. (1997), Activation of the Wnt signaling pathway: a mole...

XB-ART-16551

Dev Biol 1997 May 01;1851:82-91. doi: 10.1006/dbio.1997.8552.

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Activation of the Wnt signaling pathway: a molecular mechanism for lithium action.

Hedgepeth CM , Conrad LJ , Zhang J , Huang HC , Lee VM , Klein PS .

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Glycogen synthase kinase-3 beta (GSK-3 beta/zeste-white-3/shaggy) is a negative regulator of the wnt signaling pathway which plays a central role in the development of invertebrates and vertebrates; loss of function and dominant negative mutations in GSK-3 beta lead to activation of the wnt pathway in Drosophila and Xenopus. We now provide evidence that lithium activates downstream components of the wnt signaling pathway in vivo, leading to accumulation of beta-catenin protein. Our data indicate that this activation of the wnt pathway is a consequence of inhibition of GSK-3 beta by lithium. Using a novel assay for GSK-3 beta in oocytes, we show that lithium inhibits GSK-3 beta from species as diverse as Dictyostelium discoideum and Xenopus laevis, providing a biochemical mechanism for the action of lithium on the development of these organisms. Lithium treatment also leads to activation of an AP-1-luciferase reporter in Xenopus embryos, consistent with previous observations that GSK-3 beta inhibits c-jun activity. Activation of the wnt pathway with a dominant negative form of GSK-3 beta is inhibited by myo-inositol, similar to the previously described effect of coinjecting myo-inositol with lithium. The mechanism by which myo-inositol inhibits both dominant negative GSK-3 beta and lithium remains uncertain.

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Species referenced: Xenopus laevis
Genes referenced: ctnnb1 gsk3b jun mapt phf1

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	FIG. 1. In vitro and in vivo phosphorylation of tau protein by GSK-3b. (A) Recombinant, purified GSK-3b was incubated with [g32P]ATP in the presence of tau protein. [g-32P]ATP incorporation can be seen at 5 min (lane 2) and continues to increase over 30 min. Phosphorylated tau protein was also detected by immunostaining with PHF1, an antibody specific for tau phosphorylated at serine residues 396 and 404. GSK-3 phosphorylated tau can also be seen in this assay at 5 min (lane 2) and continues to accumulate over 30 min. (B) Stage 6 Xenopus oocytes expressing GSK-3b were injected with tau protein and samples were harvested at 5 min, 30 min, 1.5 hr, 3 hr, and 6 hr for Western blot analysis with PHF-1 antibody. In control oocytes (0GSK-3b RNA) (lanes 1– 5) tau is not phosphorylated at Ser-396 or 404 during the 6-hr incubation. However, in oocytes injected with GSK-3b RNA(lanes 6– 10) PHF1 staining can be identified at 5 min and increases in intensity over time. Equal loading of tau protein is shown in lower panels with an antibody that recognizes all forms of tau protein.
	FIG. 2. In vivo inhibition of GSK-3b by lithium. (A) Xenopus oocytes expressing GSK-3b were transferred to OR2/ containing 0 mM LiCl (lanes 3 and 5) or 20 mM LiCl (lanes 4 and 6) and were injected with the tau protein. After 2 hr oocytes were harvested for Western blot. Lane 1, no tau protein; lanes 2– 6, tau injected. GSK-3b RNA injection and lithium treatment are indicated in the figure. PHF1 and T14/T46 are as described in the legend to Fig. 1. Lanes 1, 3, and 4, injection of Xenopus GSK-3b RNA; lanes 5 and 6, injection of Dictyostelium GSK-3b RNA. (B) Selected samples from (A) were analyzed by Western blot using an anti-GSK-3 anti body, which recognizes Xenopus but not Dictyostelium GSK-3b. (C) Dose response for lithium inhibition. Oocytes were treated as in (A), but the concentration of LiCl was varied from 0 to 50 mM, as indicated in the figure. After injection of tau protein, oocyte extracts were analyzed by Western blot using PHF-1 or T14/46 antibodies as indicated.
	FIG. 3. b-Catenin stabilization by lithium. (A) Xenopus oocytes were injected with b-cateninRNA (lanes 1–4) and DN-GSK-3 RNA (lane 3), wild-type GSK-3 RNA (lane 4), or incubated in 20 mM LiCl (lane 2). Oocytes were incubated overnight in OR2/ and then harvested for Western analysis. (B) b-Catenin protein was injected into Stage 6 Xenopus oocytes and samples were taken for Western blotting at 0, 1, 2, 4, or 6 hr after incubation in OR2/ with 0 (0) or 20 mM (/) LiCl. un represents uninjected oocytes. (C) Two-cell embryos were injected with b-catenin protein and treated with lithium or control buffer as described in the text. Embryos were harvested immediately after lithium treatment or at 4 hr (blastula stage). 0 and / indicate control or lithiumtreated and un indicates uninjected embryos. inj, injected b-catenin; end, endogenous b- catenin protein. For all panels, b-catenin protein was visualized by Western blot using anti-b-catenin antibody.
	FIG. 4. Lithium activates AP-1 transcriptional activity in Xenopus embryos. (A) One-cell embryos were injected with an AP-1– luciferase reporter plasmid and cultured in the presence of 0–20 mM LiCl until stage 12 when embryos were harvested for luciferase assays. (B) Luciferase activity with AP-1–luciferase and SV40– luciferase constructs in the presence (20 mM) or absence of LiCl. The values are normalized to maximum activity for each reporter. The experiment was repeated five times and in each case AP-1 activity was stimulated in the presence of lithium (up to 145-fold), although the magnitude of the enhancement varied considerably in different batches of eggs.
	FIG. 5. Myo-inositol blocks ectopic axis induced by DN-GSK-3. Single ventral–vegetal blastomeres of 16-cell Xenopus embryos were injected with DN-GSK-3 / H2O or DN-GSK-3 / myo-inositol and were scored for formation of ectopic dorsal axis through stage 35. The panel on the left (DN-GSK-3) shows induction of a complete second axis with cement gland, head, and eyes. The panel on the right (DNGSK- 3 / myo-inositol) shows an incomplete secondary axis without clear head structures. See Table 1.