Constitutive Beta-catenin Activation Research Articles

Pde8b haploinsufficiency in mice is associated with modest adrenal defects, impaired steroidogenesis, and male infertility, unaltered by concurrent PKA or Wnt activation

PDE8B, PRKAR1A and the Wnt/β-catenin signaling are involved in endocrine disorders. However, how PDEB8B interacts with both Wnt and protein kinase A (PKA) signaling in vivo remains unknown. We created a novel Pde8b knockout mouse line (Pde8b−/−); Pde8b haploinsufficient (Pde8b+/−) mice were then crossed with mice harboring: (1) constitutive beta-catenin activation (Pde8b+/−;ΔCat) and (2) Prkar1a haploinsufficieny (Pde8b+/−;Prkar1a+/−). Adrenals and testes from mice (3-12-mo) were evaluated in addition to plasma corticosterone, aldosterone and Dkk3 concentrations, and the examination of expression of steroidogenesis-, Wnt- and cAMP/PKA-related genes. Pde8b−/− male mice were infertile with down-regulation of the Wnt/β-catenin pathway which did not change significantly in the Pde8b+/−;ΔCat mice. Prkar1a haploinsufficiency also did not change the phenotype significantly. In vitro studies showed that PDE8B knockdown upregulated the Wnt pathway and increased proliferation in CTNNB1-mutant cells, whereas it downregulated the Wnt pathway in PRKAR1A-mutant cells. These data support an overall weak, if any, role for PDE8B in adrenocortical tumorigenesis, even when co-altered with Wnt signaling or PKA upregulation; on the other hand, PDE8B appears to play a significant role in male fertility.

C22orf2 Gene (22q13.1) Translocation Downstream of Bcr Breakpoint (22q11) to the Derivative 9 Chromosome Is Associated with Reduced Expression of Its Protein, the Beta-Catenin Antagonist Chibby, in Chronic Myeloid Leukemia Progenitors

Abstract 1676Chibby is a 14.5 kDa proteins encoded by C22orf2 gene located at chromosome 22q13-1. Its major function is to antagonize beta catenin by competing for binding with Tcf/Lef transcription factors and interacting with 14-3-3 scaffolding proteins thereby promoting cytoplasmatic compartmentalization into a stable tripartite complex with beta catenin. Chibby relative vicinity to the Bcr breakpoint on chromosome 22 and its antagonistic role on beta catenin, a critical signal for self renewal and growth of CML stem cells addressed us to investigate Chibby expression in chronic myeloid leukemia (CML) and its impact on the disease prognosis. We first compared for Chibby protein expression levels in mononuclear cell fractions (MNCs) from bone marrow samples of CML patients (n. 28) at clinical diagnosis (n. 18) or in more advanced stages of the disease (n. 10) and peripheral blood aphaeresis of normal persons. A significant reduction of Chibby was detected in 14 out of 28 CML samples analyzed (p<0.01 or less) independent from the disease stage. Semi-quantitative and real-time PCR analysis revealed a significant reduction of Chibby transcript in 6/15 samples. We then investigated the expression of Chibby in the putative stem cell compartment (CD34+) isolated from bone marrow MNCs of CML patients (n.3) at diagnosis. Both Chibby protein and transcript were significantly lower (p<0.001 or less) in CD34+ cell fraction compared to the whole MNC, supporting that the down-regulation of Chibby may have a prominent role in the constitutive activation of beta catenin in an early CML progenitor compartment. Further investigation revealed that Chibby expression levels were raised in MNCs from CML patients who achieved a major molecular response under IM therapy compared with the levels seen at diagnosis. The findings support that Chibby down-regulation is indeed associated with t(9;22) translocation and is restricted to the leukemic clone. Finally, we performed FISH analysis on MNCs of all CML patients by means of 2 different probes mapping the entire region on chromosome 22q13.1 where Chibby gene is located and found that one Chibby allele is translocated to the derivative 9 chromosome still fused to upstream Bcr sequences.In conclusion, results presented here let distinguish the translocation of C22orf2 gene from chromosome 22q13-1 to the derivative chromosome 9 as a genetic lesion associated with Bcr breakpoint at 22q11 of CML. Such a genetic lesion is almost invariably associated with the down-regulation of Chibby which is likely involved in the constitutive activation of beta catenin driving the proliferative advantage of leukemic stem cells over their normal counterpart. Further investigation is required to elucidate whether Chibby down-regulation in CML is driven by transcriptional mechanisms (such as the impairment of gene regulatory sequences) or by post-transcriptional events (eventually affecting the protein interactions and stability). Disclosures:Castagnetti:Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

78: Constitutive beta-catenin activation induces hyperplasia and promotes adrenal cancer development

78: Constitutive beta-catenin activation induces hyperplasia and promotes adrenal cancer development

Constitutive β-catenin activation induces adrenal hyperplasia and promotes adrenal cancer development

Adrenocortical carcinoma is a rare but aggressive cancer with unknown aetiology. Constitutive activation of beta-catenin is the most frequent alteration in benign and malignant adrenocortical tumours in patients. Here, we show that constitutive activation of beta-catenin in the adrenal cortex of transgenic mice resulted in progressive steroidogenic and undifferentiated spindle-shaped cells hyperplasia as well as dysplasia of the cortex and medulla. Over a 17 months time course, transgenic adrenals developed malignant characteristics such as uncontrolled neovascularization and loco-regional metastatic invasion. These oncogenic events were accompanied by ectopic differentiation of glomerulosa at the expense of fasciculata cells, which caused primary hyperaldosteronism. Altogether these observations demonstrate that constitutively active beta-catenin is an adrenal oncogene which triggers benign aldosterone-secreting tumour development and promotes malignancy.

Increased skeletal VEGF enhances β-catenin activity and results in excessively ossified bones

Vascular endothelial growth factor (VEGF) and beta-catenin both act broadly in embryogenesis and adulthood, including in the skeletal and vascular systems. Increased or deregulated activity of these molecules has been linked to cancer and bone-related pathologies. By using novel mouse models to locally increase VEGF levels in the skeleton, we found that embryonic VEGF over-expression in osteo-chondroprogenitors and their progeny largely pheno-copied constitutive beta-catenin activation. Adult induction of VEGF in these cell populations dramatically increased bone mass, associated with aberrant vascularization, bone marrow fibrosis and haematological anomalies. Genetic and pharmacological interventions showed that VEGF increased bone mass through a VEGF receptor 2- and phosphatidyl inositol 3-kinase-mediated pathway inducing beta-catenin transcriptional activity in endothelial and osteoblastic cells, likely through modulation of glycogen synthase kinase 3-beta phosphorylation. These insights into the actions of VEGF in the bone and marrow environment underscore its power as pleiotropic bone anabolic agent but also warn for caution in its therapeutic use. Moreover, the finding that VEGF can modulate beta-catenin activity may have widespread physiological and clinical ramifications.

Proteomic analysis reveals a major role for contact inhibition in the terminal differentiation of hepatocytes

Hepatocytes are considered an exception of the paradigmatic inverse correlation between cell proliferation and terminal differentiation. In fact, hepatic vital functions are guaranteed by proliferating parenchymal cells during liver regeneration. However, a fine molecular characterization of the relationship between proliferation and differentiation in hepatocytes has been hampered by the lack of reliable in vivo or in vitro models. The hepatocyte terminal differentiation program was characterized in the immortalized, untransformed and differentiated hepatocytic cell line MMH, using several techniques. Particularly, two-dimensional difference gel electrophoresis combined to tandem mass spectrometry proteomic approach was used. Cell cycle and cell adhesion properties of MMH have been altered using either myc-overexpression and MEK1/2 inhibition or a constitutive active beta-catenin mutant, respectively. The hepatocyte terminal differentiation program is stimulated by the exit from the cell cycle induced by cell-cell contact. Comparative proteomic analysis of proliferating versus quiescent hepatocytes validated the importance of contact inhibition, identifying 68 differently expressed gene products, representing 49 unique proteins. Notably, enzymes involved in important liver functions such as detoxification processes, lipid metabolism, iron and vitamin A storage and secretion, anti-inflammatory response and exocytosis were found significantly up-regulated in quiescent hepatocytes. Finally, we found that: (i) cell cycle arrest induced by MEK1/2 inhibition is not sufficient to induce hepatic product expression; (ii) constitutive activation of beta-catenin counteracts the contact inhibition-induced terminal differentiation. The hepatocyte terminal differentiation program requires a quiescent state maintained by cell-cell contact through the E-cadherin/beta-catenin pathway, rather than the inhibition of proliferation.

Constitutive WNT/Beta-Catenin Signaling in Murine Sertoli Cells Disrupts Their Differentiation and Ability to Support Spermatogenesis1

Sertoli and germ cell interactions are essential for spermatogenesis and, thus, male fertility. Sertoli cells provide a specialized microenvironment for spermatogonial stem cells to divide, allowing both self-renewal and spermatogenesis. In the present study, we used mice with a conditional activated allele of the beta-catenin gene (Ctnnb1(tm1Mmt)(/+)) in Sertoli cells expressing Cre recombinase driven by the anti-Müllerian hormone (AMH; also known as Müllerian-inhibiting substance) type II receptor promoter (Amhr2(tm3(cre)Bhr)(/+)) to show that constitutively activated beta-catenin leads to their continuous proliferation and compromised differentiation. Compared to controls, Sertoli cells in mature mutant mice continue to express high levels of both AMH and glial cell-derived neurotrophic factor (GDNF), which normally are expressed only in immature Sertoli cells. We also show evidence that LiCl treatment, which activates endogenous nuclear beta-catenin activity, regulates both AMH and GDNF expression at the transcriptional level. The epididymides were devoid of sperm in the Amhr2(tm3(cre)Bhr)(/+);Ctnnb1(tm1Mmt)(/+) mice at all ages examined. We show that the mutant mice are infertile because of defective differentiation of germ cells and increased apoptosis, both of which are characteristic of GDNF overexpression in Sertoli cells. Constitutive activation of beta-catenin in Amhr2-null mice showed the same histology, suggesting that the phenotype was the result of persistent overexpression of GDNF. These results show that dysregulated wingless-related MMTV integration site/beta-catenin signaling in Sertoli cells inhibits their postnatal differentiation, resulting in increased germ cell apoptosis and infertility.

Constitutive Activation of Beta-Catenin in Uterine Stroma and Smooth Muscle Leads to the Development of Mesenchymal Tumors in Mice1

Leiomyomas and other mesenchymally derived tumors are the most common neoplasms of the female reproductive tract. Presently, very little is known about the etiology and progression of these tumors, which are the primary indication for hysterectomies. Dysregulated WNT signaling through beta-catenin is a well-established mechanism for tumorigenesis. We have developed a mouse model that expresses constitutively activated beta-catenin in uterine mesenchyme driven by the expression of Cre recombinase knocked into the Müllerian-inhibiting substance type II receptor promoter locus to investigate its effects on uterine endometrial stroma and myometrium. These mice show myometrial hyperplasia and develop mesenchymal tumors with 100% penetrance that exhibit histological and molecular characteristics of human leiomyomas and endometrial stromal sarcomas. By immunohistochemistry, we also show that both transforming growth factor beta and the mammalian target of rapamycin are induced by constitutive activation of beta-catenin. The prevalence of the tumors was greater in multiparous mice, suggesting that their development may be a hormonally driven process or that changes in uterine morphology during pregnancy and after parturition induce injury and repair mechanisms that stimulate tumorigenesis from stem/progenitor cells, which normally do not express constitutively activated beta-catenin. Additionally, adenomyosis and endometrial gland hyperplasia were occasionally observed in some mice. These results show evidence suggesting that dysregulated, stromal, and myometrial WNT/beta-catenin signaling has pleiotropic effects on uterine function and tumorigenesis.

Hematopoietic stem cell and multilineage defects generated by constitutive β-catenin activation

Gain of Wnt signaling through beta-catenin has been ascribed a critical function in the stimulation of hematopoietic stem cell self-renewal, whereas loss of beta-catenin is reportedly dispensable for hematopoiesis. Here we have used conditional mouse genetics and transplantation assays to demonstrate that constitutive activation of beta-catenin blocked multilineage differentiation, leading to the death of mice. Blood cell depletion was accompanied by failure of hematopoietic stem cells to repopulate irradiated hosts and to differentiate into mature cells. Activation of beta-catenin enforced cell cycle entry of hematopoietic stem cells, thus leading to exhaustion of the long-term stem cell pool. Our data suggest that fine-tuned Wnt stimulation is essential for hematopoiesis and is thus critical for therapeutic hematopoietic stem cell population expansion.

Essential Role of β-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.