Constitutive activation of β-catenin in ameloblasts leads to incisor enamel hypomineralization

Enamel is the hardest tissue with the highest degree of mineralization protecting the dental pulp from injury in vertebrates. The ameloblasts differentiated from ectoderm-derived epithelial cells are a single cell layer and are important for the enamel formation and mineralization. Wnt/β-catenin signaling has been proven to exert an important role in the mineralization of bone, dentin and cementum. Little was known about the regulatory mechanism of Wnt/β-catenin signaling pathway in ameloblasts during amelogenesis, especially in the mineralization of enamel. To investigate the role of β-catenin in ameloblasts, we established Amelx-Cre; β-catenin^?ex3fl/fl (CA-β-catenin) mice, which could constitutive activate β-catenin in ameloblasts. It showed the delayed mineralization and eventual hypomineralization in the incisor enamel of CA-β-catenin mice. Meanwhile, the amelogenesis-related proteinases Mmp20 and Klk4 were decreased in the incisors of CA-β-catenin mice. These data indicated that β-catenin plays an essential role in differentiation and function of ameloblasts during amelogenesis.     

Protein complexes associated with β-catenin differentially influence the differentiation profile of neonatal and adult CD8+ T cells

The canonical Wnt signaling pathway is a master cell regulator involved in CD8⁺ T cell proliferation and differentiation. In human CD8⁺ T cells, this pathway induces differentiation into memory cells or a “stem cell memory like” population, which is preferentially present in cord blood. To better understand the role of canonical Wnt signals in neonatal or adult blood, we compared the proteins associated with β-catenin, in nonstimulated and Wnt3a-stimulated human neonatal and adult naive CD8⁺ T cells. Differentially recruited proteins established different complexes in adult and neonatal cells. In the former, β-catenin-associated proteins were linked to cell signaling and immunological functions, whereas those of neonates were linked to proliferation and metabolism. Wnt3a stimulation led to the recruitment and overexpression of Wnt11 in adult cells and Wnt5a in neonatal cells, suggesting a differential connexion with planar polarity and Wnt/Ca²⁺ noncanonical pathways, respectively. The chromatin immunoprecipitation polymerase chain reaction β-catenin was recruited to a higher level on the promoters of cell renewal genes in neonatal cells and of differentiation genes in those of adults. We found a preferential association of β-catenin with CBP in neonatal cells and with p300 in the adult samples, which could be involved in a higher self-renewal capacity of the neonatal cells and memory commitment in those of adults. Altogether, our results show that different proteins associated with β-catenin during Wnt3a activation mediate a differential response of neonatal and adult human CD8⁺ T cells.     

Modulation of β-receptors as adult and neonatal cardiac myocytes progress into culture

Summary Modulation of β-adrenergic receptors and their ability to respond to β-receptor stimulation was studied in cultures of adult and neonatal rat cardiac myocytes. The radioligand iodocyanopindolol (¹²⁵I-CYP) was used to identify β-adrenoceptors on the intact cells.¹²⁵I-CYP was found to bind to the receptors in a stereospecific and saturable manner. Freshly isolated neonatal and adult myocytes both had a receptor density of approximately 50 fmol/mg protein. The number of β-receptors per milligram protein was similar during a 10-d culture period for adult myocytes but increased after a 5-d culture period for neonatal myocytes. Both cell types responded to β-receptor stimulation with isoproterenol by a twofold increase in the concentration of cAMP and this response increased with time in culture. The number of receptors as well as the response to isoproterenol was similar for neonatal myocytes cultured on laminin, collagen type I, or on uncoated culture dishes. From these data we conclude that cultured cardiac myocytes maintain functional β-receptors as they progress into culture, and the expression of β-receptors is not influenced by culture substrates. This investigation was supported by grants HL 24935 and HL 33656 from the National Institutes of Health, Bethesda, MD, and Swedish Medical Research Council grant 07466.     

Bcl-2 increases stroke-induced striatal neurogenesis in adult brains by inhibiting BMP-4 function via activation of β-catenin signaling

Our previous experiments suggest that treatment with Bcl-2 increases proliferation and differentiation of neuronal progenitors induced by ischemic injury and ameliorates neurological functional deficits after stroke. However, in addition to its traditional anti-apoptotic effect, little is known about the concrete molecular modulation mechanism. In this study, Bcl-2-expressing plasmids were injected into the lateral ventricle of rat brains immediately following a 30-min occlusion of the middle cerebral artery to determine the role of Bcl-2 in adult neurogenesis. Bcl-2 overexpression reduced ischemic infarct and astrogenesis, and enhanced ischemia-induced striatal neurogenesis. We further found that Bcl-2 increased β-catenin, a key mediator of canonical Wnt/β-catenin signaling pathway, and reduced bone morphogenetic proteins-4 (BMP-4) expression in the ipsilateral striatum following ischemia. Treatment of stroke with β-catenin siRNA (i.c.v.) showed that β-catenin siRNA antagonized Bcl-2 neuroprotection against ischemic brain injury. More interestingly, β-catenin siRNA simultaneously abolished Bcl-2-mediated reduction of BMP-4 expression and enhancement of neurogenesis in the ipsilateral striatum. This effect is independent of Noggin, the known BMP antagonist. These findings highlight a new regulatory mechanism that Bcl-2 elevates ischemia-induced striatal neurogenesis by down-regulating expression of BMP-4 via activation of the Wnt/β-catenin signaling pathway in adult rat brains.     

Forced activation of β-catenin signaling supports the transformation of hTERT-immortalized human fetal hepatocytes

Hepatocarcinogenesis is a multistep process driving the progressive transformation of normal liver cells into highly malignant derivatives. Unlimited proliferation and telomere maintenance have been recognized as prerequisites for the development of liver cancer. Moreover, recent studies identified illegitimate β-catenin signaling as relevant hit in a considerable subset of patients. To further investigate the currently not well-understood malignant evolution driven by telomerase and β-catenin, we monitored cytogenetic and phenotypic alterations in untransformed telomerase-immortalized human fetal hepatocytes following forced activation of β-catenin signaling. As expected, constitutive activation of β-catenin signaling significantly enhanced proliferation with decreasing serum dependence. Previously intact contact inhibition was almost completely eliminated. Interestingly, after several passages in cell culture, immortalized clones with dominant-positive β-catenin signaling acquired additional chromosomal aberrations, in particular translocations, anchorage-independent growth capabilities, and formed tumors in athymic nude mice. In further support for the driving role of β-catenin during hepatocarcinogenesis, improved colony growth in soft agar and accelerated tumor formation was also confirmed in Huh7 cells following stable expression of the constitutively active S33Y β-catenin mutant. Telomerase inhibition showed that short-term expansion of transformed clones was not telomerase dependent. Finally, cancer pathway profiling in derived tumors revealed upregulation of characteristic genes associated with invasion and angiogenesis. In conclusion, illegitimate activation of β-catenin signaling enhances the transformation from immortalization to malignant growth in human fetal hepatocytes. Our data functionally confirm a permissive role for β-catenin signaling in the initial phase of hepatocarcinogenesis.     

APP induces neuronal apoptosis through APP-BP1-mediated downregulation of β-catenin

Alzheimer's disease (AD) is a neurodegenerative disease associated with progressive dementia. This mini-review focuses on how the amyloid precursor protein (APP) plays a central role in AD and Down syndrome as the regulator of the APP-BP1/hUba3 activated neddylation pathway. It is argued that the physiological function of APP is to downregulate the level of beta-catenin. However, this APP function is abnormally amplified in patients with familial AD (FAD) mutations in APP and presenilins, resulting in the hyperactivation of neddylation and the decrease of beta-catenin below a threshold level. Evidence in the literature is summarized to show that dysfunction of APP in downregulating beta-catenin may underlie the mechanism of neuronal death in AD and Down syndrome.     

Oncogenic function of DACT1 in colon cancer through the regulation of β-catenin

The Wnt/β-catenin signaling pathway plays important roles in the progression of colon cancer. DACT1 has been identified as a modulator of Wnt signaling through its interaction with Dishevelled (Dvl), a central mediator of both the canonical and noncanonical Wnt pathways. However, the functions of DACT1 in the WNT/β-catenin signaling pathway remain unclear. Here, we present evidence that DACT1 is an important positive regulator in colon cancer through regulating the stability and sublocation of β-catenin. We have shown that DACT1 promotes cancer cell proliferation in vitro and tumor growth in vivo and enhances the migratory and invasive potential of colon cancer cells. Furthermore, the higher expression of DACT1 not only increases the nuclear and cytoplasmic fractions of β-catenin, but also increases its membrane-associated fraction. The overexpression of DACT1 leads to the increased accumulation of nonphosphorylated β-catenin in the cytoplasm and particularly in the nuclei. We have demonstrated that DACT1 interacts with GSK-3β and β-catenin. DACT1 stabilizes β-catenin via DACT1-induced effects on GSK-3β and directly interacts with β-catenin proteins. The level of phosphorylated GSK-3β at Ser9 is significantly increased following the elevated expression of DACT1. DACT1 mediates the subcellular localization of β-catenin via increasing the level of phosphorylated GSK-3β at Ser9 to inhibit the activity of GSK-3β. Taken together, our study identifies DACT1 as an important positive regulator in colon cancer and suggests a potential strategy for the therapeutic control of the β-catenin-dependent pathway.     

Acute activation of β2-adrenergic receptor regulates focal adhesions through βArrestin2- and p115RhoGEF protein-mediated activation of RhoA

β(2)-Adrenergic receptors (β(2)ARs) regulate cellular functions through G protein-transduced and βArrestin-transduced signals. β(2)ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that β(2)AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. β(2)ARs induce activation of RhoA, which is dependent on βArrestin2 but not G(s). βArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G(12/13)-coupled receptors. Our results show that βArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon β(2)AR activation, both βArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of β(2)ARs in cell migration. These results suggest that βArrestin2 may serve as a convergence point for non-G(12/13) and non-G(q) protein-coupled receptors to activate RhoA.     

Identification of β-catenin as a novel substrate of polo-like kinase 1

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays an important role in M phase progression by regulating various downstream substrates via phosphorylation. Here, we identified β-catenin as a novel substrate of Plk1 and determined that Ser-718 is a phosphorylation site for Plk1 by using a phospho-specific antibody that cross-reacts with Plk1-dependent phosphorylation sites. Ser-718 of β-catenin was directly phosphorylated by recombinant Plk1 in vitro, with the phosphorylation signal in cells increasing with overexpression of Plk1 and decreasing when endogenous Plk1 was depleted by small interfering RNA. The phosphorylation at Ser-718 was correlated with the cell cycle-dependent expression of Plk1 which reached a maximum in M phase. We also confirmed that there is a physical interaction between β-catenin and Plk1 using coimmunoprecipitation and a GST pull-down assay. These results demonstrate that β-catenin is a physiological substrate of Plk1 in cells, which may provide a novel insight into the role of β-catenin in M phase.     

Constitutive activation of β-catenin in odontoblasts induces aberrant pulp calcification in mouse incisors

Journal of Molecular Histology - During dentin formation, odontoblast polarization ensures that odontoblasts directionally secrete dentin matrix protein, leading to tubular dentin formation;...     

Regulation of β-catenin trafficking to the membrane in living cells

β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t_0.5 of ~ 30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t_0.5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t_0.5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.