Wnt/beta-catenin signaling is a component of osteoblastic bone cell early responses to load-bearing and requires estrogen receptor alpha

The Wnt/beta-catenin pathway has been implicated in bone cell response to their mechanical environment. This response is the origin of the mechanism by which bone cells adjust bone architecture to maintain bone strength. Osteoporosis is the most widespread failure of this mechanism. The degree of osteoporotic bone loss in men and women is related to bio-available estrogen. Here we report that in osteoblastic ROS 17/2.8 cells and primary osteoblast cultures, a single short period of dynamic mechanical strain, as well as the glycogen synthase kinase-3beta (GSK-3beta) inhibitor LiCl, increased nuclear accumulation of activated beta-catenin and stimulated TCF/LEF reporter activity. This effect was blocked by the estrogen receptor (ER) modulators ICI 182,780 and tamoxifen and was absent in primary osteoblast cultures from mice lacking ERalpha. Microarray expression data for 25,000 genes from total RNA extracted from tibiae of wild-type mice within 24 h of being loaded in vivo showed differential gene regulation between loaded and contralateral non-loaded bones of 10 genes established to be involved in the Wnt pathway. Only 2 genes were involved in loaded tibiae from mice lacking ERalpha (ERalpha(-/-)). Together these data suggest that Wnt/beta-catenin signaling contributes to bone cell early responses to mechanical strain and that its effectiveness requires ERalpha. Reduced effectiveness of bone cell responses to bone loading, associated with estrogen-related decline in ERalpha, may contribute to the failure to maintain structurally appropriate bone mass in osteoporosis in both men and women.     

Wnt/beta-catenin signaling in development and disease

A remarkable interdisciplinary effort has unraveled the WNT (Wingless and INT-1) signal transduction cascade over the last two decades. Wnt genes encode small secreted proteins that are found in all animal genomes. Wnt signaling is involved in virtually every aspect of embryonic development and also controls homeostatic self-renewal in a number of adult tissues. Germline mutations in the Wnt pathway cause several hereditary diseases, and somatic mutations are associated with cancer of the intestine and a variety of other tissues.     

Wnt/beta-catenin signaling in adipogenesis and metabolism

Adipocyte differentiation consists of a complex series of events in which scores of cellular and extracellular factors interact to transform a fibroblast-like preadipocyte into a mature, lipid-filled adipocyte. Many of the pathways influencing this process have been identified using well-characterized preadipocyte culture systems and have subsequently been confirmed in animal models. Research conducted over the past decade has established the Wnt/beta-catenin signaling pathway as an important regulator of adipocyte differentiation. While initial reports implicated activators of Wnt/beta-catenin signaling as potent inhibitors of adipogenesis, recent investigations of mesenchymal cell fate, obesity, and type 2 diabetes highlight significant additional roles for Wnt signaling in metabolism and adipocyte biology.     

CFTR and Wnt/beta-catenin signaling in lung development

Background  

Cystic fibrosis transmembrane conductance regulator (CFTR) was shown previously to modify stretch induced differentiation in the lung. The mechanism for CFTR modulation of lung development was examined by in utero gene transfer of either a sense or antisense construct to alter CFTR expression levels.     

Wnt/beta-catenin and estrogen signaling converge in vivo

Wnt and estrogen signaling represent important regulatory pathways, each controlling a wide range of biological processes. While an increasing number of observations suggest potential convergence between these pathways, no direct evidence of their functional interaction has been reported. Using human colon and breast cancer cells, we found that estrogen receptor (ER) alpha- and beta-catenin precipitated within the same immunocomplexes, reciprocally enhanced the transactivation of cognate reporter genes, and were reciprocally recruited to cognate response elements in the promoters of endogenous target genes. Using transgenic Drosophila that ectopically expressed human ERalpha alone or together with metabolically stable beta-catenin/Armadillo mutants, we demonstrated genetic interaction between these signal transducers in vivo. Thus, we present here the first direct evidence of cross-talk between Wnt and estrogen signaling pathways via functional interaction between beta-catenin and ERalpha.     

Wnt/beta-catenin signaling interacts differentially with Ihh signaling in controlling endochondral bone and synovial joint formation

Both the Wnt/beta-catenin and Ihh signaling pathways play essential roles in crucial aspects of endochondral ossification: osteoblast differentiation, chondrocyte proliferation and hypertrophy. To understand the genetic interaction between these two signaling pathways, we have inactivated the beta-catenin gene and upregulated Ihh signaling simultaneously in the same cells during endochondral skeletal development using beta-catenin and patched 1 floxed alleles. We uncovered previously unexpected roles of Ihh signaling in synovial joint formation and the essential function of Wnt/beta-catenin signaling in regulating chondrocyte survival. More importantly, we found that Wnt and Ihh signaling interact with each other in distinct ways to control osteoblast differentiation, chondrocyte proliferation, hypertrophy, survival and synovial joint formation in the developing endochondral bone. Beta-catenin is required downstream of Ihh signaling and osterix expression for osteoblast differentiation. But in chondrocyte survival, beta-catenin is required upstream of Ihh signaling to inhibit chondrocyte apoptosis. In addition, Ihh signaling can inhibit chondrocyte hypertrophy and synovial joint formation independently of beta-catenin. However, there is a strong synergistic interaction between Wnt/beta-catenin and Ihh signaling in regulating synovial joint formation.     

R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis

We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/beta-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-beta pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/beta-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/beta-catenin cascade.     

Rap2 is required for Wnt/beta-catenin signaling pathway in Xenopus early development

The Wnt/beta-catenin signaling pathway is critical for the establishment of organizer and embryonic body axis in Xenopus development. Here, we present evidence that Xenopus Rap2, a member of Ras GTPase family, is implicated in Wnt/beta-catenin signaling during the dorsoventral axis specification. Ectopic expression of XRap2 can lead to neural induction without mesoderm differentiation. XRap2 dorsalizes ventral tissues, inducing axis duplication, organizer-specific gene expression and convergent extension movements. Knockdown of XRap2 causes ventralized phenotypes including shortened body axis and defective dorsoanterior patterning, which are associated with aberrant Wnt signaling. In line with this, XRap2 depletion inhibits beta-catenin stabilization and the induction of ectopic dorsal axis and Wnt-responsive genes caused by XWnt8, Dsh or beta-catenin, but has no effect on the signaling activities of a stabilized beta-catenin. Its knockdown also disrupts the vesicular localization of Dsh, thereby inhibiting Dsh-mediated beta-catenin stabilization and the membrane recruitment and phosphorylation of Dsh by frizzled signaling. Taking together, we suggest that XRap2 is involved in Wnt/beta-catenin signaling as a modulator of the subcellular localization of Dsh.     

Flavonoids: potential Wnt/beta-catenin signaling modulators in cancer

Flavonoids are polyphenolic compounds found throughout the plant kingdom. They occur in every organ but are usually concentrated in leaves and flowers. During the last two decades, in vitro and in vivo studies demonstrated that flavonoids have inhibitory effects on human diseases through targeting of multiple cellular signaling components. Wnt/β-catenin signaling regulates proliferation, differentiation and fate specification in developmental stages and controls tissue homeostasis in adult life. For these reasons, this pathway has received great attention in the last years as potential pathway involved in distinct Human pathologies. In this review we discuss the emerging potential mechanisms for flavonoids on Wnt/β-catenin signaling in cancer and possible investigation strategies to understand flavonoids mode of action on this signaling pathway.     

Site specific bone adaptation response to mechanical loading

Over 25 million Americans suffer from osteoporosis. Bone size and strength depends both upon the level of adaptation due to physical activity (applied load), and genetics. We hypothesized that bone adaptation to loads differs among mice breeds and bone sites. Forty-five adult female mice from three inbred strains (C57BL/6 [B6], C3H/HeJ [C3], and DBA/2J [D2]) were loaded at the right tibia and ulna in vivo with non-invasive loading devices. Each loading session consisted of 99 cycles at a force range that induced approximately 2000 microstrain (microepsilon) at the mid-shaft of the tibia (2.5 to 3.5 N force) and ulna (1.5 to 2 N force). The right and left ulnae and tibiae were collected and processed using protocols for histological undecalcified cortical bone slides. Standard histomorphometry techniques were used to quantify new bone formation. The histomorphometric variables include percentage mineralizing surface (%MS), mineral apposition rate (MAR), and bone formation rate (BFR). Net loading response [right-left limb] was compared between different breeds at tibial and ulnar sites using two-way ANOVA with repeated measures (p<0.05). Significant site differences in bone adaptation response were present within each breed (p<0.005). In all the three breeds, the tibiae showed greater percentage MS, MAR and BFR than the ulna at similar in vivo load or mechanical stimulus (strain). These data suggest that the bone formation due to loading is greater in the tibiae than the ulnae. Although, no significant breed-related differences were found in response to loading, the data show greater trends in tibial bone response in B6 mice as compared to D2 and C3 mice. Our data indicate that there are site-specific skeletal differences in bone adaptation response to similar mechanical stimulus.     

Wnt/beta-catenin signaling in cancer stemness and malignant behavior

Stem cells are defined by their intrinsic capacity to self-renew and differentiate. Cancer stem cells retain both these features but have lost homeostatic mechanisms which maintain normal cell numbers. The canonical Wnt/beta-catenin signaling pathway plays a central role in modulating the delicate balance between stemness and differentiation in several adult stem cell niches such as the hair follicles in the skin, the mammary gland, and the intestinal crypt. Accordingly, constitutive Wnt signaling activation, resulting from mutations in genes encoding its downstream components, underlies tumorigenesis in these tissues. In the majority of sporadic colorectal cancer cases, the rate-limiting event is either loss of APC function or oncogenic beta-catenin mutations. However, although the presence of these initiating mutations would predict nuclear beta-catenin accumulation throughout the tumor mass, heterogeneous intracellular distributions of this key Wnt signaling molecule are observed within primary tumors and their metastases. In particular, tumor cells located at the invasive front and those migrating into the adjacent stromal tissues show nuclear beta-catenin staining. Hence, different levels of Wnt signaling activity reflect tumor heterogeneity and are likely to account for distinct cellular activities such as proliferation and epithelial-mesenchymal transitions, which prompt tumor growth and malignant behavior, respectively. Several intrinsic (cell-autonomous and/or autocrine) and extrinsic (paracrine, derived from the tumor microenvironment) factors may explain this heterogeneity of Wnt/beta-catenin signaling activity within the tumor mass.