Lrp5 functions in bone to regulate bone mass.

The human skeleton is affected by mutations in low-density lipoprotein receptor-related protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with osteocyte-specific expression of inducible Lrp5 mutations that cause high and low bone mass phenotypes in humans. We found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also induced an Lrp5 mutation in cells that form the appendicular skeleton but not in cells that form the axial skeleton; we observed that bone properties were altered in the limb but not in the spine. These data indicate that Lrp5 signaling functions locally, and they suggest that increasing LRP5 signaling in mature bone cells may be a strategy for treating human disorders associated with low bone mass, such as osteoporosis.

The Wnt co-receptor Lrp6 is required for normal mouse mammary gland development.

Canonical Wnt signals are transduced through a Frizzled receptor and either the LRP5 or LRP6 co-receptor; such signals play central roles during development and in disease. We have previously shown that Lrp5 is required for ductal stem cell activity and that loss of Lrp5 delays normal mammary development and Wnt1-induced tumorigenesis. Here we show that canonical Wnt signals through the Lrp6 co-receptor are also required for normal mouse mammary gland development. Loss of Lrp6 compromises Wnt/beta-catenin signaling and interferes with mammary placode, fat pad, and branching development during embryogenesis. Heterozygosity for an inactivating mutation in Lrp6 is associated with a reduced number of terminal end buds and branches during postnatal development. While Lrp6 is expressed in both the basal and luminal mammary epithelium during embryogenesis, Lrp6 expression later becomes restricted to cells residing in the basal epithelial layer. Interestingly, these cells also express mammary stem cell markers. In humans, increased Lrp6 expression is associated with basal-like breast cancer. Taken together, our results suggest both overlapping and specific functions for Lrp5 and Lrp6 in the mammary gland.

Wnt signaling and prostate cancer.

Canonical Wnt signaling has emerged as an important pathway that underlies the initia nottion of prostate cancer. Both human cancers and mouse models have confirmed that mutations or altered expression of components of this pathway are associated with prostate tumors. Additionally, several reports suggest that this pathway plays a key role in the establishment of skeletal metastasis. This review discusses our current knowledge of the Wnt signaling pathway in the development of prostate cancer. First, we will overview the Wnt signaling pathway to provide background for the rest of the discussion. We will then review the literature on the role of this pathway and the down notstream effector, beta-catenin, in the development and progression of prostate cancer and skeletal metastasis. We will also discuss reports that suggest that beta-catenin can directly interact with the androgen receptor to modulate its activity. These recent developments may provide insight into how tumor growth can be achieved under androgen deprivation. Finally, we speculate on how the pathway may be targeted for therapeutic treatment and what agents may be available to achieve this goal.

Inactivation of Apc in the mouse prostate causes prostate carcinoma.

Alterations of the Wnt/beta-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated Wnt/beta-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre-mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of beta-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between 12 and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of beta-catenin associated with prostate carcinoma suggests a role for beta-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of Wnt signaling with other genetic and epigenetic signals in prostate carcinogenesis.

Lifelong accumulation of bone in mice lacking Pten in osteoblasts.

Bone formation is carried out by the osteoblast, a mesenchymal cell whose lifespan and activity are regulated by growth factor signaling networks. Growth factors activate phosphatidylinositol 3-kinase (PI3K), which enhances cell survival and antagonizes apoptosis through activation of Akt/PKB. This process is negatively regulated by the Pten phosphatase, which inhibits the activity of PI3K. In this study, we investigated the effects of Akt activation in bone in vivo by conditionally disrupting the Pten gene in osteoblasts by using Cre-mediated recombination. Mice deficient in Pten in osteoblasts were of normal size but demonstrated a dramatic and progressively increasing bone mineral density throughout life. In vitro osteoblasts lacking Pten differentiated more rapidly than controls and exhibited greatly reduced apoptosis in association with markedly increased levels of phosphorylated Akt and activation of signaling pathways downstream of activated Akt. These findings support a critical role for this tumor-suppressor gene in regulating osteoblast lifespan and likely explain the skeletal abnormalities in patients carrying germ-line mutations of PTEN.

The Wnt signaling receptor Lrp5 is required for mammary ductal stem cell activity and Wnt1-induced tumorigenesis.

Canonical Wnt signaling has emerged as a critical regulatory pathway for stem cells. The association between ectopic activation of Wnt signaling and many different types of human cancer suggests that Wnt ligands can initiate tumor formation through altered regulation of stem cell populations. Here we have shown that mice deficient for the Wnt co-receptor Lrp5 are resistant to Wnt1-induced mammary tumors, which have been shown to be derived from the mammary stem/progenitor cell population. These mice exhibit a profound delay in tumorigenesis that is associated with reduced Wnt1-induced accumulation of mammary progenitor cells. In addition to the tumor resistance phenotype, loss of Lrp5 delays normal mammary development. The ductal trees of 5-week-old Lrp5-/- females have fewer terminal end buds, which are structures critical for juvenile ductal extension presumed to be rich in stem/progenitor cells. Consequently, the mature ductal tree is hypomorphic and does not completely fill the fat pad. Furthermore, Lrp5-/- ductal cells from mature females exhibit little to no stem cell activity in limiting dilution transplants. Finally, we have shown that Lrp5-/- embryos exhibit substantially impaired canonical Wnt signaling in the primitive stem cell compartment of the mammary placodes. These findings suggest that Lrp5-mediated canonical signaling is required for mammary ductal stem cell activity and for tumor development in response to oncogenic Wnt effectors.

Essential role of beta-catenin in postnatal bone acquisition.

Mutations in the Wnt co-receptor LRP5 alter bone mass in humans, but the mechanisms responsible for Wnts actions in bone are unclear. To investigate the role of the classical Wnt signaling pathway in osteogenesis, we generated mice lacking the beta-catenin or adenomatous polyposis coli (Apc) genes in osteoblasts. Loss of beta-catenin produced severe osteopenia with striking increases in osteoclasts, whereas constitutive activation of beta-catenin in the conditional Apc mutants resulted in dramatically increased bone deposition and a disappearance of osteoclasts. In vitro, osteoblasts lacking the beta-catenin gene exhibited impaired maturation and mineralization with elevated expression of the osteoclast differentiation factor, receptor activated by nuclear factor-kappaB ligand (RANKL), and diminished expression of the RANKL decoy receptor, osteoprotegerin. By contrast, Apc-deficient osteoblasts matured normally but demonstrated decreased expression of RANKL and increased osteoprotegerin. These findings suggest that Wnt/beta-catenin signaling in osteoblasts coordinates postnatal bone acquisition by controlling the differentiation and activity of both osteoblasts and osteoclasts.

Wnt-independent activation of beta-catenin mediated by a Dkk1-Fz5 fusion protein.

An XWnt8-Fz5 fusion protein synergizes with LRP6 to potently activate beta-catenin-dependent signaling. Here, we generated a fusion in which XWnt8 was fused to the N-terminus of LRP6 and show it synergizes with both Fz4 and Fz5 to potently transactivate beta-catenin-dependent Wnt signaling. Based on this, we hypothesized that the main function of Wnt is to nucleate the formation of a physical complex between LRP6 and a Frizzled. Dkk1, but not the related Dkk3, binds LRP6 and inhibits canonical Wnt signaling by blocking the interaction of Wnt and LRP6. Therefore, we reasoned that a covalent fusion of Dkk1 to Fz5 (Dkk1-Fz5) would mimic Wnt ligand by nucleating the formation of a complex containing Fz5 and LRP6, while Dkk3 (Dkk3-Fz5) would not. We found that Dkk1-Fz5, but not Dkk3-Fz5, potently synergized with LRP6 to activate signaling in a dishevelled-dependent manner.

Spectral karyotyping of sarcomas and fibroblasts derived from Ink4a/Arf-deficient mice reveals chromosomal instability in vitro.

The Ink4a/Arf locus is functionally linked to the Rb and p53 pathways through the action of its two gene products. Mouse models null for this locus show rapid onset of cancer with a preponderance of lymphomas and sarcomas. We report on a study of cell lines derived from sarcomas arising in Ink4a/Arf null mice. The cytogenetics of these lines was monitored over the course of serial passage. Results indicate that early passage cells are relatively normal. However, after multiple passages chromosomal instability becomes apparent as evidenced by increasing tetraploidy and aneuploidy, and the concomitant loss of clonality. To further evaluate the effect of Ink4a/Arf-deficiency on chromosomal stability in vitro, we isolated Ink4a/Arf deficient primary murine embryonic fibroblasts (MEFs), serially passaged them, and analyzed their chromosomal stability by spectral karyotyping (a 24-color chromosome paint-FISH technique). We found that chromosomal instability in Ink4a/Arf deficient MEFs developed with the same timing as seen in cell lines derived from Ink4a/Arf deficient sarcomas. Thus, chromosomal instability seen in Ink4a/Arf deficient tumors in vitro may be unrelated to the original phenotype of the tumor in vivo. Therefore, interpretation of cytogenetic data from cell lines derived from Ink4a/Arf deficient tumors should be done on early passage cells.

Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6.

UNLABELLED: Humans and mice lacking Lrp5 have low BMD. To evaluate whether Lrp5 and Lrp6 interact genetically to control bone or skeletal development, we created mice carrying mutations in both Lrp5 and the related gene Lrp6. We found that compound mutants had dose-dependent deficits in BMD and limb formation, suggesting functional redundancy between these two genes in bone and limb development. INTRODUCTION: Lrp5 and Lrp6 are closely related members of the low density lipoprotein receptor family and are co-receptors for Wnt ligands. While Lrp5 mutations are associated with low BMD in humans and mice, the role of Lrp6 in bone formation has not been analyzed. MATERIALS AND METHODS: To address whether Lrp5 and Lrp6 play complimentary roles in bone and skeletal development, we created mice with mutations in both genes. We inspected limbs of mice from the different genotypic classes of compound mutants to identify abnormalities. DXA and muCT were used to evaluate the effect of mutations in Lrp5 and Lrp6 on BMD and microarchitecture. RESULTS: Mice heterozygous for mutations in Lrp6 and either heterozygous or homozygous for a mutation in Lrp5 (Lrp6(+/-);Lrp5(+/-) or Lrp6(+/-);Lrp5(-/-)) display limb defects with incomplete penetrance and variable expression. DXA analysis showed that BMD decreased as mice progressively were more deficient in Lrp5 and Lrp6. Lrp6(+/-);Lrp5(-/-) mice were more severely affected than Lrp6(+/+);Lrp5(-/-) mice, whereas Lrp6(+/-);Lrp5(+/-) mice had statistically higher BMD than Lrp6(+/+);Lrp5(-/-) mice and lower BMD compared with wildtype mice and mice heterozygous for either mutation alone. CONCLUSIONS: Lrp6 and Lrp5 genetically interact in limb development in mice. Furthermore, heterozygosity for an inactivating mutation in Lrp6 further reduces BMD in both male and female mice lacking Lrp5.

A novel set of Wnt-Frizzled fusion proteins identifies receptor components that activate beta -catenin-dependent signaling.

Wnt proteins initiate the canonical (beta-catenin-regulated) signaling cascade by binding to seven-transmembrane spanning receptors of the Frizzled (Fz) family together with the coreceptors LRP5 and -6, members of the low density lipoprotein receptor-related protein family (LRP). Several reports have shown physical and functional associations between various Wnt, LRP, and Frizzled molecules; however, the underlying mechanisms for selectivity remain poorly understood. We present data on a novel set of Wnt-Fz fusion constructs that are useful for elucidating mechanisms of Wnt signal transduction specificity in both Xenopus embryos and 293T cells. In 293T cells, coexpression of several Wnt-Fz fusion proteins with LRP6, but not LRP5, significantly activated a Wnt-responsive promoter, Optimized TOPFlash. Interestingly, Wnt proteins from both the Wnt1 and Wnt5A classes, when fused to the same Frizzled, can synergize with LRP6 to activate signaling and induce secondary axes in Xenopus embryos. However, when several Wnt-Fz constructs containing different Frizzled molecules were tested, it was found that all Frizzled molecules are not equivalent in their ability to activate the canonical Wnt pathway in this context. The data suggest that the distinction between the two Wnt classes lies not in intrinsic differences in the molecules but via the Frizzled molecules with which they interact.