Lrp5 and Lrp6 exert overlapping functions in osteoblasts during postnatal bone acquisition

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

An osteocalcin-deficient mouse strain without endocrine abnormalities

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/2p.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/ 2pPro25fs17Ter 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

Modulation of β-Catenin Signaling by Glucagon Receptor Activation

The glucagon receptor (GCGR) is a member of the class B G protein–coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA) pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin–mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R) and glucagon-like peptide 1 (GLP-1R) receptors. Since low-density-lipoprotein receptor–related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter–mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1) or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations

Wnt/β-catenin Signaling in Normal and Cancer Stem Cells

The ability of Wnt ligands to initiate a signaling cascade that results in cytoplasmic stabilization of, and nuclear localization of, β-catenin underlies their ability to regulate progenitor cell differentiation. In this review, we will summarize the current knowledge of the mechanisms underlying Wnt/β-catenin signaling and how the pathway regulates normal differentiation of stem cells in the intestine, mammary gland, and prostate. We will also discuss how dysregulation of the pathway is associated with putative cancer stem cells and the potential therapeutic implications of regulating Wnt signaling

Inhibiting WNT secretion reduces high bone mass caused by Sost loss-of-function or gain-of-function mutations in Lrp5.

Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the activity of the pathway in the skeleton, cause high bone mass in human subjects and mouse models. Increased bone mass is often accompanied by severe headaches from increased intracranial pressure, which can lead to fatality and loss of vision or hearing due to the entrapment of cranial nerves. In addition, progressive forehead bossing and mandibular overgrowth occur in almost all subjects. Treatments that would provide symptomatic relief in these subjects are limited. Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the frizzled receptor. Chemical inhibition of porcupine is a highly selective method of Wnt signaling inhibition. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling, including homozygosity for loss-of-function in Sost, which models sclerosteosis, and two strains of mice carrying different point mutations in Lrp5 (equivalent to human G171V and A214V), at 3 months of age with porcupine inhibitors for 5–6 weeks. Treatment significantly reduced both trabecular and cortical bone mass in all three models. This demonstrates that porcupine inhibition is potentially therapeutic for symptomatic relief in subjects who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models

Loss of Lrp6 impairs osteoblast differentiation.

<p>(A) Lrp6 mRNA levels in primary calvarial osteoblasts were assessed by qPCR during 14 days of osteogenic differentiation. (B) Lrp6 and Lrp5 mRNA levels were assessed by qPCR in calvarial osteoblasts isolated from Lrp6 floxed mice and infected with adenovirus expressing GFP (control) or Cre recombinase. (C) Axin2 mRNA levels were examined in control and ΔLrp6 osteoblasts in cells treated with 100 ng/ml Wnt-3a. (D) Cell proliferation was assessed by BrdU incorporation. Osteogenic (E) and osteoclastogenic markers (F) were examined by qPCR after 14 days of differentiation in osteogenic media. (G) Mineralized nodule formation was assessed by staining for alkaline phosphatase (AP), von Kossa (VK) and Alizarin red (ARS) after 14 days of differentiation. *p<0.05.</p

Trabecular and cortical bone acquisition is reduced in ΔLrp6 mice.

<p>(A) PCR analysis of Cre-mediated recombination of the Lrp6^flox allele in ΔLrp6 mice. (B) qPCR analysis of Lrp6 mRNA levels in the femur of control and ΔLrp6 mice. (C) Whole-bone mineral density assessed by DXA at 6months of age (n = 16–26 mice). (D) Representative microCT images of bone structure in the distal femur of control and ΔLrp6 mice at 8, 16, and 24 weeks of age. (E) Quantification of trabecular bone volume per tissue volume (BV/TV) in the distal femur. (F) Quantification of trabecular numbers (Tb.N) in the distal femur. (G) Representative microCT images of cortical bone structure at the femoral mid-diaphysis in 24 weeks old control and ΔLrp6 mice. (H) Cortical tissue area (Tt.Ar). (I) Cortical bone area per tissue area (Ct.Ar/Tt.Ar). (J) Cortical thickness (Ct. Th). (K) Polar moment of inertia (pMOI). For microCT analyses, n = 5–8mice/genotype. *p<0.05.</p

Lrp5 acts in the late stages of osteoblast differentiation.

<p>(A) Lrp5 mRNA levels in primary calvarial osteoblasts were assessed by qPCR during 14 days of osteogenic differentiation. (B) Lrp5 and Lrp6 mRNA levels were assessed by qPCR in calvarial osteoblasts isolated from Lrp5 floxed mice and infected with adenovirus expressing GFP (control) or Cre recombinase. (C) Axin2 mRNA levels were examined in control and ΔLrp5 osteoblasts in cells treated with 100 ng/ml Wnt-3a. (D) Cell proliferation was assessed by BrdU incorporation. Osteogenic (E) and osteoclastogenic markers (F) were examined by qPCR after 14 days of differentiation in osteogenic media. (G) Mineralized nodule formation was assessed by staining for alkaline phosphatase (AP), von Kossa (VK) and Alizarin red (ARS) after 14 days of differentiation. *p<0.05.</p

ΔLrp5 mice develop an osteopenic phenotype.

<p>(A) PCR analysis of Cre-mediated recombination of the Lrp5^flox allele in ΔLrp5 mice. Note the recombination (upper panel) is only evident in skeletal tissue. (B) qPCR analysis of Lrp5 mRNA levels in the femur of control and ΔLrp5 mice. (C) Whole-bone mineral density assessed by DXA at 6months of age (n = 16–18 mice). (D) Representative microCT images of bone structure in the distal femur of control and ΔLrp5 mice at 8, 16, and 24 weeks of age. (E) Quantification of trabecular bone volume per tissue volume (BV/TV) in the distal femur. (F) Quantification of trabecular numbers (Tb.N) in the distal femur. (G) Representative microCT images of cortical bone structure at the femoral mid-diaphysis in 24 weeks old control and ΔLrp5 mice. (H) Cortical tissue area (Tt.Ar). (I) Cortical bone area per tissue area (Ct.Ar/Tt.Ar). (J) Cortical thickness (Ct. Th). (K) Polar moment of inertia (pMOI). For microCT analyses, n = 5–7mice/genotype. *p<0.05.</p