Liver-specific β-catenin knockout (β-Catenin-LKO) mice have revealed an essential role of β-catenin in metabolic zonation where it regulates pericentral gene expression and in initiating liver regeneration (LR) after partial hepatectomy (PH), by regulating expression of Cyclin-D1. However what regulates β-catenin activity in these events remains an enigma. Here, we investigate to what extent β-catenin activation is Wnt-signaling dependent and the potential cell source of Wnts. We studied liver-specific Lrp5/6 KO (Lrp-LKO) mice where Wnt-signaling was abolished in hepatocytes while the β-catenin gene remained intact. Intriguingly, like β-catenin-LKO mice, Lrp-LKO exhibited a defect in metabolic zonation observed as lack of glutamine synthetase (GS), Cyp1a2 and Cyp2e1. Lrp-LKO also displayed a significant delay in initiation of LR due to absence of β-catenin-TCF4 association and lack of Cyclin-D1. To address the source of Wnt proteins in liver, we investigated conditional Wntless (Wls) KO mice, which lacked ability to secrete Wnts from either liver epithelial cells (Wls-LKO), or macrophages including Kupffer cells (Wls-MKO), or endothelial cells (Wls-EKO). While Wls-EKO was embryonic lethal precluding further analysis in adult hepatic homeostasis and growth, Wls-LKO and Wls-MKO were viable but did not show any defect in hepatic zonation. Wls-LKO showed normal initiation of LR, however Wls-MKO showed a significant but temporal deficit in LR that was associated with decreased β-catenin-TCF4 association and diminished Cyclin-D1 expression. Conclusion Wnt-signaling is the major upstream effector of β-catenin activity in pericentral hepatocytes and during LR. Hepatocytes, cholangiocytes or macrophages are not the source of Wnts in regulating hepatic zonation. However, Kupffer cells are a major contributing source of Wnt secretion necessary for β-catenin activation during LR.

The canonical Wnt signaling pathway is critical for skeletal development and maintenance, but the precise roles of the individual Wnt co-receptors, Lrp5 and Lrp6, that enable Wnt signals to be transmitted in osteoblasts remain controversial. In these studies, we used Cre-loxP recombination, in which Cre-expression is driven by the human osteocalcin promoter, to determine the individual contributions of Lrp5 and Lrp6 in postnatal bone acquisition and osteoblast function. Mice selectively lacking either Lrp5 or Lrp6 in mature osteoblasts were born at the expected Mendelian frequency but demonstrated significant reductions in whole-body bone mineral density. Bone architecture measured by microCT revealed that Lrp6 mutant mice failed to accumulate normal amounts of trabecular bone. By contrast, Lrp5 mutants had normal trabecular bone volume at 8 weeks of age, but with age, these mice also exhibited trabecular bone loss. Both mutants also exhibited significant alterations in cortical bone structure. In vitro differentiation was impaired in both Lrp5 and Lrp6 null osteoblasts as indexed by alkaline phosphatase and Alizarin red staining, but the defect was more pronounced in Lrp6 mutant cells. Mice lacking both Wnt co-receptors developed severe osteopenia similar to that observed previously in mice lacking β-catenin in osteoblasts. Likewise, calvarial cells doubly deficient for Lrp5 and Lrp6 failed to form osteoblasts when cultured in osteogenic media, but instead attained a chondrocyte-like phenotype. These results indicate that expression of both Lrp5 and Lrp6 are required within mature osteoblasts for normal postnatal bone development.

Osteocalcin (OCN), the most abundant noncollagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc -) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double-knockout (dko) allele (Bglap/2 p.Pro25fs17Ter ) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full-length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their serum. FTIR imaging of cortical bone in these homozygous knockout animals finds alterations in the collagen maturity and carbonate to phosphate ratio in the cortical bone, compared with wild-type littermates. However, μCT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc − allele, serum glucose levels and male fertility in the OCN-deficient mice with the Bglap/ 2 pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Possible explanations include the effects of each mutated allele on the transcription of neighboring genes, or differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double-knockout strain to the Jackson Laboratories for academic distribution.

Dysregulated Wnt signaling is associated with the pathogenesis of cancers, fibrosis, and vascular diseases. Inhibition of Wnt signaling has shown efficacy in various pre-clinical models of these disorders. One of the key challenges in developing targeted anti-cancer drugs is to balance efficacy with on-target toxicity. Given the crucial role Wnts play in the differentiation of osteoblasts and osteoclasts, acute inhibition of Wnt signaling is likely to affect bone homeostasis. In this study, we evaluated the skeletal effect of small molecule inhibitor of an o-acyl transferase porcupine (PORCN) that prevents Wnt signaling by blocking the secretion of all Wnts. Micro-computed tomography and histomorphometric evaluation revealed that the bones of mice treated with two structurally distinct PORCN inhibitors LGK974 and ETC-1922159 (ETC-159) had loss-of-bone volume and density within 4 weeks of exposure. This decreased bone mass was associated with a significant increase in adipocytes within the bone marrow. Notably, simultaneous administration of a clinically approved anti-resorptive, alendronate, a member of the bisphosphonate family, mitigated loss-of-bone mass seen upon ETC-159 treatment by regulating activity of osteoclasts and blocking accumulation of bone marrow adipocytes. Our results support the addition of bone protective agents when treating patients with PORCN inhibitors. Mitigation of bone toxicity can extend the therapeutic utility of Wnt pathway inhibitors.

Cross-talk between the canonical Wnt and mammalian target of rapamycin (mTOR) signaling pathways occurs at multiple levels in the cell and likely contributes to the oncogenic effects of these pathways in human cancer. To gain more insight into the interplay between Wnt and mTOR signaling in salivary gland tumorigenesis, we developed a mouse model in which both pathways are constitutively activated by the conditional inactivation of the Apc and Pten tumor suppressor genes. Loss of either Apc or Pten alone did not cause tumor development. However, deletion of both genes resulted in the formation of salivary gland tumors with 100% penetrance and short latency that showed a remarkable morphologic similarity to human acinic cell carcinoma. Treatment of tumor-bearing mice using the mTOR inhibitor rapamycin led to complete regression of tumors, indicating that tumor growth was dependent on continued mTOR signaling. Importantly, we found that human salivary gland acinic cell carcinomas also express markers of activated mTOR signaling. Together, these results suggest that aberrant activation of mTOR signaling plays a pivotal role in acinar cell neoplasia of the salivary gland. Because rapamycin analogues are approved for treating other types of human malignancies, our findings suggest that rapamycin therapy should be evaluated for treating patients with salivary gland acinic cell carcinoma.