The role of pleiotrophin and β-catenin in fetal lung development

Mammalian lung development is a complex biological process, which is temporally and spatially regulated by growth factors, hormones, and extracellular matrix proteins. Abnormal changes of these molecules often lead to impaired lung development, and thus pulmonary diseases. Epithelial-mesenchymal interactions are crucial for fetal lung development. This paper reviews two interconnected pathways, pleiotrophin and Wnt/β-catenin, which are involved in fibroblast and epithelial cell communication during fetal lung development.     

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;...     

Phenotype of single hepatocytes expressing an activated version of β-catenin in liver of transgenic mice

The gene CTNNB1 encoding β-catenin is mutated in about 30% of hepatocellular carcinoma, generally often combined with other genetic alterations. In transgenic mice, it has been shown that activation of β-catenin in more than 70% of all hepatocytes causes immediate proliferation leading to hepatomegaly. In this study we established a novel mouse model where β-catenin is activated only in individual, dispersed hepatocytes. Hepatocyte-specific expression of activated point-mutated β-catenin (human β-catenin^S33Y) was established using the Cre/loxP system. Expression of several downstream targets of β-catenin signaling such as glutamine synthetase and several cytochrome P450 isoforms was confirmed by immunostaining. Only a minor portion of hepatocytes expressed the β-catenin^S33Y transgene, which were mainly positioned as dispersed individual cells within the normal liver parenchyma. The hepatocytes with activated β-catenin did not show increased proliferation and the mice did not develop hepatomegaly. In conclusion, activated β-catenin in single hepatocytes induces a gene expression pattern in hepatocytes which is similar to that of Ctnnb1-mutated mouse liver tumors, but is apparently not sufficient to induce increased cell proliferation. Therefore, onset of proliferation seems to require concomitant activation of β-catenin in clusters of hepatocytes, suggesting a role of cell–cell communication in this process.     

Roles of β-catenin in somitogenesis in rat embryos

Summary We studied the roles of β-catenin in somitogenesis using immunostaining and antisense experiments in rat embryos. High levels of β-catenin appeared transiently in the developing rat somites. Initially, β-catenin accumulation was observed in the core cells of presomitic cell aggregates and then in the lumen of epithelial vesicles. Subsequently, it was confined to the dermomyotomes and their lumen and then the myotomes. High levels of cyclin D1 were observed in the core cells, in the lumen of epithelial vesicles, in myotomes, and in mesenchymal sclerotomes. When embryos were cultured in medium supplemented with β-catenin antisense oligodeoxynucleotide (ODN), the accumulation of β-catenin, but not of cyclin D1, in the nascent somites and dermomyotomes was suppressed, while the number of somites was the same as that observed in control embryos. The number of myosin-positive somites and the amount of myosin per somite in embryos treated with the antisense ODN were lower than those in controls. These results suggested that β-catenin promotes development of myotomal cells during somitogenesis. The function of β-catenin in the development of myotomes may not be correlated to cyclin D1.     

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.     

Regulation of β-catenin stabilization in human platelets

The Wnt/β-catenin pathway controls developmental processes and homeostasis; however, abnormal activation of this pathway has been linked to several human diseases. Recent reports have demonstrated regulation of platelet function by canonical and non-canonical Wnt signalling. Platelet aggregation plays a crucial role in haemostasis and thrombosis. Here we report for the first time that, induction of sustained aggregation of platelets by a strong agonist in the presence of calcium was associated with nearly complete proteolysis of β-catenin, which was abrogated upon depletion of calcium from platelet suspension. β-catenin cleavage was disallowed in absence of aggregation, thus implicating integrin α_IIbβ₃ engagement in β-catenin proteolysis. Degradation of β-catenin was blocked partially by inhibitors of either proteasome or calpain and completely when cells were exposed to both the inhibitors. Protein kinase C inhibition, too, abolished β-catenin degradation. Thus activities of proteasome, calpain and protein kinase C regulate stabilization of β-catenin in aggregated human platelets.     

Suppression of insulin-like3 receptor reveals the role of β-catenin and Notch signaling in gubernaculum development

During male development, the testes move from a high intraabdominal position and descend into the scrotum. The gubernaculum, an inguinoscrotal ligament connecting the testis to the lower abdomen, is believed to play a critical role in this process. The first stage of testicular descent is controlled by insulin like3 hormone (INSL3), produced in testicular Leydig cells. Deletion of Insl3 or its receptor, Rxfp2, in mice causes cryptorchidism. We produced Cre/loxP regulated shRNA transgenic mice targeting RXFP2 expression. We have shown that the transgene was able to reduce Rxfp2 gene expression and thus behaved as a hypomorphic allele of Rxfp2. Variable degrees of uni- and bilateral cryptorchidism was detected in males with the activated shRNA transgene on an Rxfp2+/- background. Conditional suppression of Rxfp2 in the gubernaculum led to cryptorchidism. Gene expression analysis of a mutant cremasteric sac using Illumina microarrays indicated abnormal expression of a significant number of genes in Wnt/β-catenin and Notch pathways. We have demonstrated profound changes in the expression pattern of β-catenin, Notch1, desmin, and androgen receptor (AR), in Rxfp2-/- male embryos, indicating the role of INSL3 in proliferation, differentiation, and survival of specific cellular components of the gubernaculum. We have shown that INSL3/RXFP2 signaling is essential for myogenic differentiation and maintenance of AR-positive cells in the gubernaculum. Males with the deletion of β-catenin or Notch1 in the gubernacular ligament demonstrated abnormal development. Our data indicates that β-catenin and Notch pathways are potential targets of INSL3 signaling during gubernacular development.     

PKM2 depletion induces the compensation of glutaminolysis through β-catenin/c-Myc pathway in tumor cells

The metabolic activity in cancer cells primarily rely on aerobic glycolysis. Besides glycolysis, some tumor cells also exhibit excessive addition to glutamine, which constitutes an advantage for tumor growth. M2-type pyruvate kinase (PKM2) plays a pivotal role in sustaining aerobic glycolysis, pentose phosphate pathway and serine synthesis pathway. However, the participation of PKM2 in glutaminolysis is little to be known. Here we demonstrated that PKM2 depletion could provoke glutamine metabolism by enhancing the β-catenin signaling pathway and consequently promoting its downstream c-Myc-mediated glutamine metabolism in colon cancer cells. Treatment with 2-deoxy-d-glucose (2-DG), a glycolytic inhibitor, got consistent results with the above. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities, plays a critical role in regulating β-catenin. Moreover, we found that overexpression of PKM2 negatively regulated β-catenin through miR-200a. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon glucose metabolism impaired.     

Disruption of β-arrestins blocks glucocorticoid receptor and severely retards lung and liver development in mice

In this study, the role of β-arrestin 1 and β-arrestin 2 in fetal lung and liver development was examined using Arrb1(-/-)Arrb2(-/-) mouse embryos. β-Arrestin 1/2 dual-null mice died shortly after birth and morphological examination revealed an obvious pulmonary hypoplasia and severe hepatic impairment. Western blot analysis demonstrated that GR protein levels in Arrb1(-/-)Arrb2(-/-) lung and liver tissues were significantly decreased compared to wild type embryos. Expression of GR proteins was confirmed in the nuclei of type II pneumocytes of 18.5 day embryos (E18.5) by immunofluorescence. The production of hepatic glucose and mRNA level of gluconeogenic enzymes were dramatically reduced in E18.5 Arrb1(-/-)Arrb2(-/-) liver. These results suggest that GR is an important downstream effector of the β-arrestin signaling pathway involved in regulation of lung and liver development. However, no obvious changes in GR expression following in vitro modulation of β-arrestin 1/2 indicated the existence of an indirect regulatory relationship between GR and the β-arrestin signaling pathway.     

The cytoplasmic domain of MUC1 induces hyperplasia in the mammary gland and correlates with nuclear accumulation of β-catenin

MUC1 is an oncoprotein that is overexpressed in up to 90% of breast carcinomas. A previous in vitro study by our group demonstrated that the cytoplasmic domain of MUC1 (MUC1-CD), the minimal functional unit of MUC1, contributes to the malignant phenotype in cells by binding directly to β-catenin and protecting β-catenin from GSK3β-induced degradation. To understand the in vivo role of MUC1-CD in breast development, we generated a MUC1-CD transgenic mouse model under the control of the MMTV promoter in a C57BL/6J background, which is more resistant to breast tumor. We show that the expression of MUC1-CD in luminal epithelial cells of the mammary gland induced a hyperplasia phenotype characterized by the development of hyper-branching and extensive lobuloalveoli in transgenic mice. In addition to this hyperplasia, there was a marked increase in cellular proliferation in the mouse mammary gland. We further show that MUC1-CD induces nuclear localization of β-catenin, which is associated with a significant increase of β-catenin activity, as shown by the elevated expression of cyclin D1 and c-Myc in MMTV-MUC1-CD mice. Consistent with this finding, we observed that overexpression of MUC1-C is associated with β-catenin nuclear localization in tumor tissues and increased expression of Cyclin D1 and c-Myc in breast carcinoma specimens. Collectively, our data indicate a critical role for MUC1-CD in the development of mammary gland preneoplasia and tumorigenesis, suggesting MUC1-CD as a potential target for the diagnosis and chemoprevention of human breast cancer.     

Activation of Wnt-β-catenin pathway in basal–parabasal layers of normal cervical epithelium comparable during development of uterine cervical carcinoma

In this study, importance of Wnt-β-catenin pathway in the development of uterine cervical carcinoma was evaluated. For this purpose, the profiles (expression/methylation/deletion) of β-catenin, p-β-catenin (Y654), Wnt3a, and APC were studied in disease free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of primary tumors (n = 70), CIN (n = 28), CACX (n = 102) samples, and two CACX cell lines (HeLa and SiHa). Immunohistochemical analysis revealed high/medium (74–95%) expression of β-catenin/p-β-catenin (Y654) and Wnt3a and low expression (23–26%) of APC in proliferating basal–parabasal layers contrary to differentiated spinous layer in normal cervix irrespective of HPV16 infection. The expression profile of the genes in the basal–parabasal layers did not change significantly during development of CACX. High (66%) promoter methylation of APC was seen in basal–parabasal layers and the cervical lesions (42–69%), unlike in spinous layers (25%). The promoter methylation status of APC was validated by in vitro demethylation experiments using 5-aza-dC in CACX cell lines. However, additional deletion of APC was significantly increased from CIN (12%) to stage I/II (40%) and became comparable in stage III/IV (48%) of the tumor. Patients with alterations (deletion/methylation) of APC and high/medium expression of Wnt3a/β-catenin/p-β-catenin (Y654) showed significantly poor survival. Thus our data indicate that cumulative effect of Wnt3a overexpression and APC inactivation are needed for overexpression of β-catenin during the development of CACX.     

Biological maturation and β-adrenergic effectors: development of β-adrenergic receptors in rabbit heart

Summary The -adrenergic receptor, transduction processes and catalytic activity of the adenylate cyclase enzyme complex have been investigated in rabbit heart at different stages of biological maturation. The binding of [³H]-dihydroalprenolol to a washed membrane preparation isolated from rabbit ventricular muscle was used to characterize -adrenergic receptors. Significant age-related differences were noted in -receptor affinity (K_d) and density (RD) of neonatal and adult animals; the adult K_d was 3.7-fold greater and the RD 2-fold higher than the neonates. No significant differences in these parameters were detected among the 27-day old fetus and the 1- and 7-day old neonates. Age-dependent differences in agonist isoproterenol affinity for the receptor were not observed in contrast to the significant changes in antagonist (DHA) affinity.Age-related changes in receptor affinity were also quantitated by determining the inhibitory potency of alprenolol on isoproterenol stimulated adenylate cyclase enzyme activity. A decreased affinity of the -adrenergic receptor for alprenolol in the adult heart was indicated by a 3.7-fold greater K_i for the adult than the 1-day old neonate. Ontogenic variations in the coupling efficiency between the receptor and catalytic components of the adenylate cyclase complex were also evaluated. The K_d of the -adrenergic receptor for isoproterenol and the EC50 for adenylate cyclase stimulation were determined under similar conditions. The corresponding coupling index (K_d/EC50) was found to be 2.4-fold greater in the 1-day old neonate than adult, suggesting that for a given percentage increase in adenylate cyclase activity, a lower percentage of -adrenergic receptor sites need be occupied in the neonate. These data extend previous studies (29) and indicate all components of the rabbit heart adenylate cyclase enzyme complex (i.e., the -adrenergic receptor, the GTP-dependent transduction event, and the catalytic subunit) exhibit significant developmental changes.     

Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis

Human β-1,4-galactosyltransferase (β-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from β-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the β-1,4-GalT I-VI genes revealed that the expression of the β-1,4-GalT V gene in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells are a half and null when compared to that of B4galt5 ( +/+ )-derived MEF cells without altering the expression levels of other β-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When β-1,4-GalT activities were determined towards GlcNAcβ-S-pNP, no significant difference in its specific activity was obtained among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[(3)H]Gal, Lac-Cer synthase activity in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells decreased to 41% and 11% of that of B4galt5 ( +/+ )-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5 ( -/- ) -derived MEF cells decreased remarkably when compared with those of B4galt5 ( +/+ )-derived MEF cells. These results indicate that murine β-1,4-GalT V is involved in Lac-Cer biosynthesis.     

Inhibition of cyclooxygenase-2 enhanced intestinal epithelial homeostasis via suppressing β-catenin signalling pathway in experimental liver fibrosis

The intestinal barrier dysfunction is crucial for the development of liver fibrosis but can be disturbed by intestinal chronic inflammation characterized with cyclooxygenase-2 (COX-2) expression. This study focused on the unknown mechanism by which COX-2 regulates intestinal epithelial homeostasis in liver fibrosis. The animal models of liver fibrosis induced with TAA were established in rats and in intestinal epithelial–specific COX-2 knockout mice. The impacts of COX-2 on intestinal epithelial homeostasis via suppressing β-catenin signalling pathway were verified pharmacologically and genetically in vivo. A similar assumption was tested in Ls174T cells with goblet cell phenotype in vitro. Firstly, disruption of intestinal epithelial homeostasis in cirrhotic rats was ameliorated by celecoxib, a selective COX-2 inhibitor. Then, β-catenin signalling pathway in cirrhotic rats was associated with the activation of COX-2. Furthermore, intestinal epithelial–specific COX-2 knockout could suppress β-catenin signalling pathway and restore the disruption of ileal epithelial homeostasis in cirrhotic mice. Moreover, the effect of COX-2/PGE2 was dependent on the β-catenin signalling pathway in Ls174T cells. Therefore, inhibition of COX-2 may enhance intestinal epithelial homeostasis via suppression of the β-catenin signalling pathway in liver fibrosis.     

Role of β-catenin in post-meiotic male germ cell differentiation

Though roles of β-catenin signaling during testis development have been well established, relatively little is known about its role in postnatal testicular physiology. Even less is known about its role in post-meiotic germ cell development and differentiation. Here, we report that β-catenin is highly expressed in post-meiotic germ cells and plays an important role during spermiogenesis in mice. Spermatid-specific deletion of β-catenin resulted in significantly reduced sperm count, increased germ cell apoptosis and impaired fertility. In addition, ultrastructural studies show that the loss of β-catenin in post-meiotic germ cells led to acrosomal defects, anomalous release of immature spermatids and disruption of adherens junctions between Sertoli cells and elongating spermatids (apical ectoplasmic specialization; ES). These defects are likely due to altered expression of several genes reportedly involved in Sertoli cell-germ cell adhesion and germ cell differentiation, as revealed by gene expression analysis. Taken together, our results suggest that β-catenin is an important molecular link that integrates Sertoli cell-germ cell adhesion with the signaling events essential for post-meiotic germ cell development and maturation. Since β-catenin is also highly expressed in the Sertoli cells, we propose that binding of germ cell β-catenin complex to β-catenin complex on Sertoli cell at the apical ES surface triggers a signaling cascade that regulates post-meiotic germ cell differentiation.     

Requirement of Wnt/β-catenin signaling in pronephric kidney development

The pronephric kidney controls water and electrolyte balance during early fish and amphibian embryogenesis. Many Wnt signaling components have been implicated in kidney development. Specifically, in Xenopus pronephric development as well as the murine metanephroi, the secreted glycoprotein Wnt-4 has been shown to be essential for renal tubule formation. Despite the importance of Wnt signals in kidney organogenesis, little is known of the definitive downstream signaling pathway(s) that mediate their effects. Here we report that inhibition of Wnt/β-catenin signaling within the pronephric field of Xenopus results in significant losses to kidney epithelial tubulogenesis with little or no effect on adjoining axis or somite development. We find that the requirement for Wnt/β-catenin signaling extends throughout the pronephric primordium and is essential for the development of proximal and distal tubules of the pronephros as well as for the development of the duct and glomus. Although less pronounced than effects upon later pronephric tubule differentiation, inhibition of the Wnt/β-catenin pathway decreased expression of early pronephric mesenchymal markers indicating it is also needed in early pronephric patterning. We find that upstream inhibition of Wnt/β-catenin signals in zebrafish likewise reduces pronephric epithelial tubulogenesis. We also find that exogenous activation of Wnt/β-catenin signaling within the Xenopus pronephric field results in significant tubulogenic losses. Together, we propose Wnt/β-catenin signaling is required for pronephric tubule, duct and glomus formation in Xenopus laevis, and this requirement is conserved in zebrafish pronephric tubule formation.