K-Ras and β-catenin mutations cooperate with Fgfr3 mutations in mice to promote tumorigenesis in the skin and lung, but not in the bladder

The human fibroblast growth factor receptor 3 (FGFR3) gene is frequently mutated in superficial urothelial cell carcinoma (UCC). To test the functional significance of FGFR3 activating mutations as a ‘driver’ of UCC, we targeted the expression of mutated Fgfr3 to the murine urothelium using Cre-loxP recombination driven by the uroplakin II promoter. The introduction of the Fgfr3 mutations resulted in no obvious effect on tumorigenesis up to 18 months of age. Furthermore, even when the Fgfr3 mutations were introduced together with K-Ras or β-catenin (Ctnnb1) activating mutations, no urothelial dysplasia or UCC was observed. Interestingly, however, owing to a sporadic ectopic Cre recombinase expression in the skin and lung of these mice, Fgfr3 mutation caused papilloma and promoted lung tumorigenesis in cooperation with K-Ras and β-catenin activation, respectively. These results indicate that activation of FGFR3 can cooperate with other mutations to drive tumorigenesis in a context-dependent manner, and support the hypothesis that activation of FGFR3 signaling contributes to human cancer

Canonical Wnt Signaling Induces BMP-4 to Specify Slow Myofibrogenesis of Fetal Myoblasts

Background The Wnts are secreted proteins that play important roles in skeletal myogenesis, muscle fiber type diversification, neuromuscular junction formation and muscle stem cell function. How Wnt proteins orchestrate such diverse activities remains poorly understood. Canonical Wnt signaling stabilizes β-catenin, which subsequently translocate to the nucleus to activate the transcription of TCF/LEF family genes. Methods We employed TCF-reporter mice and performed analysis of embryos and of muscle groups. We further isolated fetal myoblasts and performed cell and molecular analyses. Results We found that canonical Wnt signaling is strongly activated during fetal myogenesis and weakly activated in adult muscles limited to the slow myofibers. Muscle-specific transgenic expression of a stabilized β-catenin protein led to increased oxidative myofibers and reduced muscle mass, suggesting that canonical Wnt signaling promotes slow fiber types and inhibits myogenesis. By TCF-luciferase reporter assay, we identified Wnt-1 and Wnt-3a as potent activators of canonical Wnt signaling in myogenic progenitors. Consistent with in vivo data, constitutive overexpression of Wnt-1 or Wnt-3a inhibited the proliferation of both C2C12 and primary myoblasts. Surprisingly, Wnt-1 and Wnt-3a overexpression up-regulated BMP-4, and inhibition of BMP-4 by shRNA or recombinant Noggin protein rescued the myogenic inhibitory effect of Wnt-1 and Wnt-3a. Importantly, Wnt-3a or BMP-4 recombinant proteins promoted slow myosin heavy chain expression during myogenic differentiation of fetal myoblasts. Conclusions These results demonstrate a novel interaction between canonical Wnt and BMP signaling that induces myogenic differentiation towards slow muscle phenotype

The Wnt/β-Catenin Pathway Interacts Differentially with PTHrP Signaling to Control Chondrocyte Hypertrophy and Final Maturation

Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through β-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate PTHrP expression. In addition, Wnt/β-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/β-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent

Optic cup and facial patterning defects in ocular ectoderm β-catenin gain-of-function mice

BACKGROUND: The canonical Wnt signaling pathway has a number of critical functions during embryonic development and, when activated aberrantly, in the genesis of cancer. Current evidence suggests that during eye development, regulation of Wnt signaling is critical for patterning the surface ectoderm that will contribute to multiple components of the eye. Wnt signaling loss-of-function experiments show that a region of periocular ectoderm will form ectopic lentoid bodies unless the Wnt pathway modifies its fate towards other structures. Consistent with this, Wnt signaling gain of function in the ocular region ectoderm results in a suppression of lens fate. RESULTS: Here we demonstrate that ectoderm-specific Wnt signaling gain-of-function embryos exhibit additional defects besides those noted in the lens. There are profound facial defects including a foreshortened snout, malformation of the nasal region, and clefting of the epidermis along the ocular-nasal axis. Furthermore, despite the restriction of Wnt pathway gain-of-function to the surface ectoderm, the optic cup is inappropriately patterned and ultimately forms a highly convoluted, disorganized array of epithelium with the characteristics of retina and retinal pigmented epithelium. CONCLUSION: We suggest that activation of the Wnt pathway in surface ectoderm may disrupt the normal exchange of signals between the presumptive lens and retina that coordinate development of a functional eye

Early Embryonic Lethality Caused by Targeted Disruption of the Mouse Thioredoxin Gene

AbstractThioredoxins belong to a widely distributed group of small proteins with strong reducing activities mediated by a consensus redox-active dithiol (Cys-Gly-Pro-Cys). Thioredoxin was first isolated as a hydrogen donor for enzymatic synthesis of deoxyribonucleotides by ribonucleotide reductase inEscherichia coli.Recent studies have revealed a variety of roles that thioredoxin plays in transcription, growth control, and immune function. In this report, we describe the phenotype of mice carrying a targeted disruption of the thioredoxin gene (Txn). Heterozygotes are viable, fertile, and appear normal. In contrast, homozygous mutants die shortly after implantation, and the concepti were resorbed prior to gastrulation. When preimplantation embryos were placed in culture, the inner cell mass cells of the homozygous embryos failed to proliferate. These results indicate thatTxnexpression is essential for early differentiation and morphogenesis of the mouse embryo

Immunology and Inflammation

In the thymus, the thymic epithelium provides a microenvironment essential for the development of functionally competent and self-tolerant T cells. Previous findings showed that modulation of Wnt/β-catenin signaling in mouse thymic epithelial cells (TECs) disrupts embryonic thymus organogenesis. However, the role of β-catenin in TECs for postnatal T-cell development remains to be elucidated. Here, we analyzed gain-of-function (GOF) and loss-of-function (LOF) of β-catenin highly specific in mouse TECs. We found that GOF of β-catenin in TECs results in severe thymic dysplasia and T-cell deficiency beginning from the embryonic period. By contrast, LOF of β-catenin in TECs reduces the number of cortical TECs and thymocytes modestly and only postnatally. These results indicate that fine-tuning of β-catenin expression within a permissive range is required for TECs to generate an optimal microenvironment to support postnatal T-cell development

Muscarinic Agonist-Mediated Heterologous Desensitization in Isolated Ileum Requires Activation of Both Muscarinic M2 and M3 Receptors

We investigated the subtypes of the muscarinic receptor mediating short-term heterologous desensitization in the isolated ileum. Treatment of the ileum from C57BL/6 mice with acetylcholine (30 μM) for 20 min caused a subsequent decrease in contractile sensitivity to both prostaglandin F2α (PGF2α) and the muscarinic agonist, oxotremorine-M. This subsensitivity was characterized by 7- and 3-fold increases in the EC50 values of the agonists, respectively, with no significant effect on the maximal response. The subsensitivity to PGF2α was prevented in both M2 and M3 muscarinic receptor knockout mice. Similarly, the subsensitivity to oxotremorine-M was prevented in M2 knockout mice. Acetylcholine-mediated desensitization of histamine-induced contractions in the guinea pig ileum was inhibited by both M2- and M3-selective muscarinic antagonists with high potency, although careful analysis of the data suggested behavior more consistent with an M2 antagonistic profile. Modeling studies showed that the competitive antagonism of response contingent upon activation of two receptor subtypes should exhibit a pharmacological profile similar to that of the least sensitive signaling pathway. Our results demonstrate that muscarinic agonist-mediated short-term heterologous desensitization of intestinal smooth muscle is contingent upon activation of both M2 and M3 muscarinic receptors and that activation of either receptor by itself is insufficient to cause desensitization

Promoter methylation regulates cyclooxygenase expression in breast cancer

INTRODUCTION: Overexpression of cyclooxygenase (COX-2) is commonly observed in human cancers. In a murine model of metastatic breast cancer, we observed that COX-2 expression and enzyme activity were associated with enhanced tumorigenic and metastatic potential. In contrast to the high COX-2 expression in metastatic tumors, transplantation of poorly tumorigenic tumor cell lines to syngeneic mice results in less COX-2 expression and less COX-2 activity in vivo. Aberrant CpG island methylation, and subsequent silencing of the COX-2 promoter, has been observed in human cancer cell lines and in some human tumors of the gastrointestinal tract. METHODS: Using bisulfite modification and a methylation-specific PCR, we examined the methylation status of the COX-2 promoter in a series of four closely-related murine mammary tumors differing in COX-2 expression and metastatic potential. RESULTS: We showed that line 410, which does not express COX-2 in vivo, exhibited evidence of promoter methylation. Interestingly, the metastatic counterpart of this cell (line 410.4) displayed only the unmethylated COX-2 promoter, as did two additional cell lines (lines 66.1 and 67). The methylation patterns observed in vitro were maintained when these murine mammary tumor lines were transplanted to syngeneic mice. Treatment with the DNA demethylating agent 5-aza-deoxycytidine increased COX-2 mRNA, increased protein and increased enzyme activity (prostaglandin synthesis). CONCLUSIONS: These results indicate that COX-2 promoter methylation may be one mechanism by which tumor cells regulate COX-2 expression. Upregulation of COX-2 expression in closely related metastatic lesions versus nonmetastatic lesions may represent a shift towards the unmethylated phenotype

β-Catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system

Abstractβ-Catenin is an essential component of the canonical Wnt signaling system that controls decisive steps in development. We employed here two conditional β-catenin mutant alleles to alter β-catenin signaling in the central nervous system of mice: one allele to ablate β-catenin and the second allele to express a constitutively active β-catenin. The tissue mass of the spinal cord and brain is reduced after ablation of β-catenin, and the neuronal precursor population is not maintained. In contrast, the spinal cord and brain of mice that express activated β-catenin is much enlarged in mass, and the neuronal precursor population is increased in size. β-Catenin signals are thus essential for the maintenance of proliferation of neuronal progenitors, controlling the size of the progenitor pool, and impinging on the decision of neuronal progenitors to proliferate or to differentiate