Regulation of YAP and Wnt signaling by the endosomal protein MAMDC4.

Maintenance of the intestinal epithelium requires constant self-renewal and regeneration. Tight regulation of proliferation and differentiation of intestinal stem cells within the crypt region is critical to maintaining homeostasis. The transcriptional co-factors ß-catenin and YAP are required for proliferation during normal homeostasis as well as intestinal regeneration after injury: aberrant signaling activity results in over proliferation and tumorigenesis. Although both YAP and ß-catenin activity are controlled along canonical pathways, it is becoming increasingly clear that non-canonical regulation of these transcriptional regulators plays a role in fine tuning their activity. We have shown previously that MAMDC4 (Endotubin, AEGP), an integral membrane protein present in endosomes, regulates both YAP and ß-catenin activity in kidney epithelial cells and in the developing intestinal epithelium. Here we show that MAMDC4 interacts with members of the signalosome and mediates cross-talk between YAP and ß-catenin. Interestingly, this cross-talk occurs through a non-canonical pathway involving interactions between AMOT:YAP and AMOT:ß-catenin.

"Did I just do that?"-Six-month-olds learn the contingency between their vocalizations and a visual reward in 5 minutes.

It has been shown that infants can increase or modify a motorically available behavior such as sucking, kicking, arm waving, etc., in response to a positive visual reinforcement (e.g., DeCasper & Fifer, 1980; Millar, 1990; Rochat & Striano, 1999; Rovee-Collier, 1997; Watson & Ramey, 1972). We tested infants to determine if they would also change their vocal behavior in response to contingent feedback, which lacks the social, emotional, and auditory modeling typical of parent-child interaction. Here, we show that in a single five-minute session infants increase the rate of their vocalizations in order to control the appearance of colorful shapes on an iPad screen. This is the first experimental study to demonstrate that infants can rapidly learn to increase their vocalizations, when given positive reinforcement with no social element. This work sets the foundations for future studies into the causal relationship between the number of early vocalizations and the onset of words. In addition, there are potential clinical applications for reinforcing vocal practice in infant populations who are at risk for poor language skills.

Proliferation in the developing intestine is regulated by the endosomal protein Endotubin.

During postnatal intestinal development, the intestinal epithelium is highly proliferative, and this proliferation is regulated by signaling in the intervillous and crypt regions. This signaling is primarily mediated by Wnt, and requires membrane trafficking. However, the mechanisms by which membrane trafficking regulates signaling during this developmental phase are largely unknown. Endotubin (EDTB, MAMDC4) is an endosomal protein that is highly expressed in the apical endocytic complex (AEC) of villus enterocytes during fetal and postnatal development, and knockout of EDTB results in defective formation of the AEC and giant lysosome. Further, knockout of EDTB in cell lines results in decreased proliferation. However, the role of EDTB in proliferation during the development of the intestine is unknown. Using Villin-CreERT2 in EDTBfl/fl mice, we deleted EDTB in the intestine in the early postnatal period, or in enteroids in vitro after isolation of intervillous cells. Loss of EDTB results in decreased proliferation in the developing intestinal epithelium and decreased ability to form enteroids. EDTB is present in cells that contain the stem cell markers LGR5 and OLFM4, indicating that it is expressed in the proliferative compartment. Further, using immunoblot analysis and TCF/LEF-GFP mice as a reporter of Wnt activity, we find that knockout of EDTB results in decreased Wnt signaling. Our results show that EDTB is essential for normal proliferation during the early stages of intestinal development and suggest that this effect is through modulation of Wnt signaling.

Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis. METHODS: We disrupted the histocompatibility 2, class II antigen A, beta 1 gene (H2-Ab1) in IECs of C57BL/6 mice (I-AbΔIEC) or Rag1-/- mice (Rag1-/-I-AbΔIEC); we used I-AbWT mice as controls. Colitis was induced by administration of DSS, transfer of CD4+CD45RBhi T cells, or infection with Citrobacter rodentium. Colon tissues were collected and analyzed by histology, immunofluorescence, xMAP, and reverse-transcription polymerase chain reaction and organoids were generated. Microbiota (total and immunoglobulin [Ig]A-coated) in intestinal samples were analyzed by16S amplicon profiling. IgA+CD138+ plasma cells from Peyer's patches and lamina propria were analyzed by flow cytometry and IgA repertoire was determined by next-generation sequencing. RESULTS: Mice with IEC-specific loss of MHCII (I-AbΔIEC mice) developed less severe DSS- or T-cell transfer-induced colitis than control mice. Intestinal tissues from I-AbΔIEC mice had a lower proportion of IgA-coated bacteria compared with control mice, and a reduced luminal concentration of secretory IgA (SIgA) following infection with C rodentium. There was no significant difference in the mucosal IgA repertoire of I-AbΔIEC vs control mice, but opsonization of cultured C rodentium by SIgA isolated from I-AbΔIEC mice was 50% lower than that of SIgA from mAbWT mice. Fifty percent of I-AbΔIEC mice died after infection with C rodentium, compared with none of the control mice. We observed a transient but significant expansion of the pathogen in the feces of I-AbΔIEC mice compared with I-AbWT mice. CONCLUSIONS: In mice with DSS or T-cell-induced colitis, loss of MHCII from IECs reduces but does not eliminate mucosal inflammation. However, in mice with C rodentium-induced colitis, loss of MHCII reduces bacterial clearance by decreasing binding of IgA to commensal and pathogenic bacteria.

The Endosomal Protein Endotubin Is Required for Enterocyte Differentiation.

BACKGROUND & AIMS: During late embryonic development and through weaning, enterocytes of the ileum are highly endocytic. Defects in endocytosis and trafficking are implicated in neonatal disease, however, the mechanisms regulating trafficking during the developmental period are incompletely understood. The apical endosomal protein endotubin (EDTB) is highly expressed in the late embryonic and neonatal ileum. In epithelial cells in vitro, EDTB regulates both trafficking of tight junction proteins and proliferation through modulation of YAP activity. However, EDTB function during the endocytic stage of development of the intestine is unknown. METHODS: By using Villin-CreERT2, we induced knockout of EDTB during late gestation and analyzed the impact on endocytic compartments and enterocyte structure in neonates using immunofluorescence, immunocytochemistry, and transmission electron microscopy. RESULTS: Deletion of the apical endosomal protein EDTB in the small intestine during development impairs enterocyte morphogenesis, including loss of the apical endocytic complex, defective formation of the lysosomal compartment, and some cells had large microvillus-rich inclusions similar to those observed in microvillus inclusion disease. There also was a decrease in apical endocytosis and mislocalization of proteins involved in apical trafficking. CONCLUSIONS: Our results show that EDTB-mediated trafficking within the epithelial cells of the developing ileum is important for maintenance of endocytic compartments and enterocyte integrity during early stages of gut development.

Endosomal regulation of contact inhibition through the AMOT:YAP pathway.

Contact-mediated inhibition of cell proliferation is an essential part of organ growth control; the transcription coactivator Yes-associated protein (YAP) plays a pivotal role in this process. In addition to phosphorylation-dependent regulation of YAP, the integral membrane protein angiomotin (AMOT) and AMOT family members control YAP through direct binding. Here we report that regulation of YAP activity occurs at the endosomal membrane through a dynamic interaction of AMOT with an endosomal integral membrane protein, endotubin (EDTB). EDTB interacts with both AMOT and occludin and preferentially associates with occludin in confluent cells but with AMOT family members in subconfluent cells. EDTB competes with YAP for binding to AMOT proteins in subconfluent cells. Overexpression of the cytoplasmic domain or full-length EDTB induces translocation of YAP to the nucleus, an overgrowth phenotype, and growth in soft agar. This increase in proliferation is dependent upon YAP activity and is complemented by overexpression of p130-AMOT. Furthermore, overexpression of EDTB inhibits the AMOT:YAP interaction. EDTB and AMOT have a greater association in subconfluent cells compared with confluent cells, and this association is regulated at the endosomal membrane. These data provide a link between the trafficking of tight junction proteins through endosomes and contact-inhibition-regulated cell growth.

Upregulation of the apelin-APJ pathway promotes neointima formation in the carotid ligation model in mouse.

AIMS: To investigate apelin-APJ (angiotensin receptor-like 1) signalling in vascular remodelling, we have examined the pathophysiological response to carotid ligation in apelin knockout mice. METHODS AND RESULTS: Apelin null animals compared with wild-type mice had significantly decreased neointimal lesion area (1.17 +/- 0.17 vs. 3.33 +/- 1.04 x 10(4) microm(2), P < 0.05) and intima/media ratio (0.81 +/- 0.23 vs. 1.49 +/- 0.44, P < 0.05), averaged over four sites 0.5-2 mm from the ligation. Exogenous apelin infusion rescued the apelin-KO phenotype, promoting neointima formation in the null animals. Apelin null animals showed decreased smooth muscle positive area in the neointima (82.3 +/- 2.4 vs. 63.9 +/- 8.4, P < 0.05), and a smaller percentage BrdU positive cells in the neointima and media (11.06 +/- 1.00 vs. 6.53 +/- 0.86, P < 0.05). Apelin mRNA expression increased initially (5.2-fold, P < 0.01) followed by increased apelin receptor expression (10.1-fold, P < 0.05) in the ligated artery. Cytochemistry studies localized apelin expression to luminal endothelial cells and apelin receptor upregulation to smooth muscle cells (SMC) in the media and neointima. In vitro experiments with cultured rat aortic SMC revealed that apelin stimulation increased migration. In contrast to the increased expression of apelin and apelin receptor in carotid remodelling, expression was not upregulated in the apoE high fat model, and correlated with the known disease-inhibitory effect in this model. CONCLUSION: These data suggest that increased apelin receptor expression by SMC provides a paracrine pathway in injured vessels that allows endothelial-derived apelin to stimulate their division and migration into the neointima.

A putative role for apelin in the etiology of obesity.

Apelin, the endogenous ligand of the G protein-coupled APJ receptor has been shown to promote tumor angiogenesis. However, the effect of apelin on inducing angiogenesis in adipose tissue has not been investigated. In this review, we propose a putative role for apelin in promoting angiogenesis in adipose tissue. We further propose that targeting adipose tissue vasculature by blocking apelin signaling with anti-apelin antibodies will lead not only to inhibition of angiogenesis in adipose tissue but also to decreased adiposity.

Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo.

The peptide growth factor apelin is the high affinity ligand for the G-protein-coupled receptor APJ. During embryonic development of mouse and frog, APJ receptor is expressed at high levels in endothelial precursor cells and in nascent vascular structures. Characterization of Xenopus apelin shows that the sequence of the bioactive region of the peptide is perfectly conserved between frogs and mammals. Embryonic expression studies indicate that apelin is expressed in, or immediately adjacent to, a subset of the developing vascular structures, particularly the intersegmental vessels. Experimental inhibition of either apelin or APJ expression, using antisense morpholino oligos, results in elimination or disruption of intersegmental vessels in a majority of embryos. In gain of function experiments, apelin peptide is a potent angiogenic factor when tested using two in vivo angiogenesis assays, the frog embryo and the chicken chorioallantoic membrane. Furthermore, studies using the mouse brain microvascular cell line bEnd.3 show that apelin acts as a mitogenic, chemotactic and anti-apoptotic agent for endothelial cells in culture. Finally, we show that, similar to a number of other angiogenic factors, expression of the apelin gene is increased under conditions of hypoxia. Taken together, these studies indicate that apelin is required for normal vascular development in the frog embryo and has properties consistent with a role during normal and pathological angiogenesis.

Detection of a large-scale mass redistribution in the terrestrial system since 1998.

Earth's dynamic oblateness (J2) had been undergoing a decrease, according to space geodetic observations over the past 25 years, until around 1998, when it switched quite suddenly to an increasing trend that has continued to the present. The secular decrease in J2 resulted primarily from the postglacial rebound in the mantle. The present increase, whose geophysical cause(s) are uncertain, thus signifies a large change in global mass distribution with a J2 effect that considerably overshadows that of mantle rebound.