Lrp5-independent activation of Wnt signaling by lithium chloride increases bone formation and bone mass in mice

Abstract

One of the well characterized cell biologic actions of lithium is the inhibition of glycogen synthase kinase-3beta and the consequent activation of canonical Wnt signaling. Because deficient Wnt signaling has been implicated in disorders of reduced bone mass, we tested whether lithium could improve bone mass in mice. We gavage-fed lithium chloride to 8-week-old mice from three different strains (Lrp5(-/-), SAMP6, and C57BL/6) and assessed the effect on bone metabolism after 4 weeks of therapy. Lrp5(-/-) mice lack the Wnt coreceptor low-density lipoprotein receptor-related protein 5 and have markedly reduced bone mass. Lithium, which is predicted to act downstream of this receptor, restored bone metabolism and bone mass to near wild-type levels in these mice. SAMP6 mice have accelerated osteoporosis due to inadequate osteoblast renewal. Lithium significantly improved bone mass in these mice and in wild-type C57BL/6 mice. We found that lithium activated canonical Wnt signaling in cultured calvarial osteoblasts from Lrp5(-/-) mice ex vivo and that lithium-treated mice had increased expression of Wnt-responsive genes in their bone marrow cells in vivo. These data lead us to conclude that lithium enhances bone formation and improves bone mass in mice and that it may do so via activation of the canonical Wnt pathway. Lithium has been used safely and effectively for over half a century in the treatment of bipolar illness. Prospective studies in patients receiving lithium should determine whether it also improves bone mass in humans.

Figures

Fig. 1.

Bone phenotype of Lrp5-/- (-/-) mice. (a) Photomicrographs of coronal sections through proximal tibias of 12-week-old Lrp5+/+ (+/+), Lrp5+/- (+/-), and Lrp5-/- (-/-) mice stained with Toluidine blue. Note the decreased numbers of trabeculae in heterozygous and homozygous mutant mice. The inset numbers represent trabecular BV/TV based on analysis of 6-12 mice of each genotype. (b) Fluorescent photomicrograph of bone trabecula from 12-week-old Lrp5+/+ (+/+), Lrp5+/- (+/-), and Lrp5-/- (-/-) mice after calcein double labeling. Inset numbers represent mineral MAR based on analysis of nine Lrp5+/+ (+/+) mice and six Lrp5-/- (-/-) mice. (c) Three-dimensional microCT reconstruction of proximal tibias from 4-week-old Lrp5+/+ and Lrp5-/-, showing reduced trabecular bone volume in the mutant mouse. *, Significant difference from wild type at P < 0.05; **, significant difference from wild type at P < 0.01.

Fig. 2.

LiCl effects in cells derived from Lrp5+/+ and Lrp5-/- mice. (a) Fold-induction of firefly luciferase normalized to renilla luciferase in osteoblasts harvested from Lrp5+/+ and Lrp5-/- mice. Cells were transiently transfected with Topflash and Renilla reporters and then cultured in the presence of combinations of 20 mM LiCl, 50 ng/ml Wnt3a, and 50 ng/ml Dkk1. Wnt3a induces luciferase expression in Lrp5+/+, but not in Lrp5-/- cells. LiCl induces expression in both genotypes and is not inhibited by Dkk1. (b) Percent apoptotic calvarial osteoblasts harvested from Lrp5+/+ and Lrp5-/- mice and cultured with or without 20 mM LiCl. LiCl reduces apoptosis in Lrp5-/- osteoblasts. (c) Phase contrast micrographs of calvaria cells harvested from Lrp5+/+ mice and cultured in medium containing either 20 mM NaCl or 20 mM LiCl. In contrast to cells cultured in NaCl that can differentiate along the adipocyte lineage (arrows), cells cultured in LiCl do not become adipocytes. (d) Fold increase in mRNA for the Wnt responsive genes Ahr, Nkd2, and Axin2. Total RNA were prepared from bone marrow cells recovered from LiCl-treated and from vehicle-treated mice. The expression level of selected genes was determined by real-time PCR and normalized on the basis of Gapdh expression. Data are presented as fold increase in mRNA expression level in LiCl treated to control vehicle treated cells. (e and f) ALP activity and ColIα1 gene expression in embryonic murine mesenchymal stem cells derived from from Lrp5+/+ and Lrp5-/- mice and cultured in the presence of either Wnt3a (50 ng/ml) or LiCl (20 mM). After 4-day stimulation, ALP activity was assessed in cell lysates. Total RNA was also extracted and ColIα1 expression was analyzed by real-time PCR and normalized on the basis of Gapdh expression. Both Wnt3a and LiCl were able to increase the activity of ALP and ColIα1 gene expression.

Fig. 3.

Lithium therapy increases bone mass and decreases bone marrow adiposity. (a-f and i-k) Von Kossa-stained coronal sections through proximal tibias of individual 12-week-old mice. Note the general increase in number of bone trabeculae (arrows) in LiCl mice. hPTH-treated SAMP6 mouse serves as a positive control. (a-c) Lrp5-/- mice that were gavage fed vehicle. (d-f) Lrp5-/- mice that were gavage fed LiCl. (g and h) Three-dimensional microCT reconstruction of proximal tibias from Lrp5-/- treated with vehicle (g) and treated with LiCl (h), showing increased trabecular bone volume in response to lithium. (i) SAMP6 mouse gavage fed vehicle. (j) SAMP6 mouse gavage fed LiCl. (k) SAMP6 mouse treated with hPTH. (l-p) H&E-stained trabecular bone from the proximal tibias of individual 12-week-old mice looking at marrow adiposity. (l) Lrp5-/- mouse that was gavage fed vehicle. (m) Lrp5-/- mouse that was gavage fed LiCl. (n) SAMP6 mouse that was gavage fed vehicle. (o) SAMP6 mouse that was gavage fed LiCl. (p) SAMP6 mouse that received s.c. hPTH.