Makorin-2 is a neurogenesis inhibitor downstream of phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signal

Makorin-2 belongs to the makorin RING zinc finger gene family, which encodes putative ribonucleoproteins. Here we cloned the Xenopus makorin-2 (mkrn2) and characterized its function in Xenopus neurogenesis. Forced overexpression of mkrn2 produced tadpoles with dorso-posterior deficiencies and small-head/short-tail phenotype, whereas knockdown of mkrn2 by morpholino antisense oligonucleotides induced double axis in tadpoles. In Xenopus animal cap explant assay, mkrn2 inhibited activin, and retinoic acid induced animal cap neuralization, as evident from the suppression of a pan neural marker, neural cell adhesion molecule. Surprisingly, the anti-neurogenic activity of mkrn2 is independent of the two major neurogenesis signaling cascades, BMP-4 and Wnt8 pathways. Instead, mkrn2 works specifically through the phosphatidylinositol 3-kinase (PI3K) and Akt-mediated neurogenesis pathway. Overexpression of mkrn2 completely abrogated constitutively active PI3K- and Akt-induced, but not dominant negative glycogen synthase kinase-3beta (GSK-3beta)-induced, neural cell adhesion molecule expression, indicating that mkrn2 acts downstream of PI3K and Akt and upstream of GSK-3beta. Moreover, mkrn2 up-regulated the mRNA and protein levels of GSK-3beta. These results revealed for the first time the important role of mkrn2 as a new player in PI3K/Akt-mediated neurogenesis during Xenopus embryonic development.     

Signaling network crosstalk in human pluripotent cells: a Smad2/3-regulated switch that controls the balance between self-renewal and differentiation

A general mechanism for how intracellular signaling pathways in human pluripotent cells are coordinated and how they maintain self-renewal remain to be elucidated. In this report, we describe a signaling mechanism where PI3K/Akt activity maintains self-renewal by restraining prodifferentiation signaling through suppression of the Raf/Mek/Erk and canonical Wnt signaling pathways. When active, PI3K/Akt establishes conditions where Activin A/Smad2,3 performs a pro-self-renewal function by activating target genes, including Nanog. When PI3K/Akt signaling is low, Wnt effectors are activated and function in conjunction with Smad2,3 to promote differentiation. The switch in Smad2,3 activity after inactivation of PI3K/Akt requires the activation of canonical Wnt signaling by Erk, which targets Gsk3β. In sum, we define a signaling framework that converges on Smad2,3 and determines its ability to regulate the balance between alternative cell states. This signaling paradigm has far-reaching implications for cell fate decisions during early embryonic development.     

Rap2 is required for Wnt/beta-catenin signaling pathway in Xenopus early development

The Wnt/beta-catenin signaling pathway is critical for the establishment of organizer and embryonic body axis in Xenopus development. Here, we present evidence that Xenopus Rap2, a member of Ras GTPase family, is implicated in Wnt/beta-catenin signaling during the dorsoventral axis specification. Ectopic expression of XRap2 can lead to neural induction without mesoderm differentiation. XRap2 dorsalizes ventral tissues, inducing axis duplication, organizer-specific gene expression and convergent extension movements. Knockdown of XRap2 causes ventralized phenotypes including shortened body axis and defective dorsoanterior patterning, which are associated with aberrant Wnt signaling. In line with this, XRap2 depletion inhibits beta-catenin stabilization and the induction of ectopic dorsal axis and Wnt-responsive genes caused by XWnt8, Dsh or beta-catenin, but has no effect on the signaling activities of a stabilized beta-catenin. Its knockdown also disrupts the vesicular localization of Dsh, thereby inhibiting Dsh-mediated beta-catenin stabilization and the membrane recruitment and phosphorylation of Dsh by frizzled signaling. Taking together, we suggest that XRap2 is involved in Wnt/beta-catenin signaling as a modulator of the subcellular localization of Dsh.     

Akt induces osteoclast differentiation through regulating the GSK3β/NFATc1 signaling cascade

SHIP is an SH2-containing inositol-5-phosphatase expressed in hematopoietic cells. It hydrolyzes the PI3K product PI(3,4,5)P(3) and blunts the PI3K-initiated signaling pathway. Although the PI3K/Akt pathway has been shown to be important for osteoclastogenesis, the molecular events involved in osteoclast differentiation have not been revealed. We demonstrate that Akt induces osteoclast differentiation through regulating the GSK3β/NFATc1 signaling cascade. Inhibition of the PI3K by LY294002 reduces formation of osteoclasts and attenuates the expression of NFATc1, but not that of c-Fos. Conversely, overexpression of Akt in bone marrow-derived macrophages (BMMs) strongly induced NFATc1 expression without affecting c-Fos expression, suggesting that PI3K/Akt-mediated NFATc1 induction is independent of c-Fos during RANKL-induced osteoclastogenesis. In addition, we found that overexpression of Akt enhances formation of an inactive form of GSK3β (phospho-GSK3β) and nuclear localization of NFATc1, and that overexpression of a constitutively active form of GSK3β attenuates osteoclast formation through downregulation of NFATc1. Furthermore, BMMs from SHIP knockout mice show the increased expression levels of phospho-Akt and phospho-GSK3β, as well as the enhanced osteoclastogenesis, compared with wild type. However, overexpression of a constitutively active form of GSK3β attenuates RANKL-induced osteoclast differentiation from SHIP-deficient BMMs. Our data suggest that the PI3K/Akt/GSK3β/NFATc1 signaling axis plays an important role in RANKL-induced osteoclastogenesis.     

Roles of ADAM13-regulated Wnt activity in early Xenopus eye development

Pericellular proteolysis by ADAM family metalloproteinases has been widely implicated in cell signaling and development. We recently found that Xenopus ADAM13, an ADAM metalloproteinase, is required for activation of canonical Wnt signaling during cranial neural crest (CNC) induction by regulating a novel crosstalk between Wnt and ephrin B (EfnB) signaling pathways (Wei et al., 2010b). In the present study we show that the metalloproteinase activity of ADAM13 also plays important roles in eye development in Xenopus tropicalis. Knockdown of ADAM13 results in reduced expression of eye field markers pax6 and rx1, as well as that of the pan-neural marker sox2. Activation of canonical Wnt signaling or inhibition of forward EfnB signaling rescues the eye defects caused by loss of ADAM13, suggesting that ADAM13 functions through regulation of the EfnB-Wnt pathway interaction. Downstream of Wnt, the head inducer Cerberus was identified as an effector that mediates ADAM13 function in early eye field formation. Furthermore, ectopic expression of the Wnt target gene snail2 restores cerberus expression and rescues the eye defects caused by ADAM13 knockdown. Together these data suggest an important role of ADAM13-regulated Wnt activity in eye development in Xenopus.     

Regulation of survivin by PI3K/Akt/p70S6K1 pathway

PI3K activation is commonly observed in many human cancer cells. Survivin expression is elevated in cancer cells, and induced by some growth factors through PI3K activation. However, it is not clear whether PI3K activation is sufficient to induce survivin expression. To investigate the role of PI3K pathway in the regulation of survivin, we expressed an active form of PI3K, v-P3k in chicken embryonic fibroblast cells (CEF), and found that overexpression of PI3K-induced survivin mRNA expression. Forced expression of wild-type but not mutant tumor suppressor PTEN in CEF decreased survivin mRNA levels. PI3K regulates survivin expression through Akt activation. To further investigate downstream target of PI3K and Akt in regulating the expression of survivin mRNA, we found that PI3K and Akt-induced p70S6K1 activation and that overexpression of p70S6K1 alone was sufficient to induce survivin expression. The treatment of CEF cells by rapamycin decreased the survivin mRNA expression. This result demonstrated that p70S6K1 is an important target downstream of PI3K and Akt in regulating suvivin mRNA expression. The knockdown of survivin mRNA expression by its specific siRNA induced apoptosis of cancer cells when the cells were treated with LY294002 or taxol. Taken together, these results demonstrated that PI3K/Akt/p70S6K1 pathway is essential for regulating survivin mRNA expression.     

PI3K/Akt pathway regulates retinoic acid‐induced Hox gene expression in F9 cells

Retinoic acid (RA), the most potent natural form of vitamin A, is a key morphogen in vertebrate development and a potent regulator of both adult and embryonic cell differentiation. Specifically, RA regulates clustered Hox gene expression during embryogenesis and is required to establish the anteroposterior body plan. The PI3K/Akt pathway was also reported to play an essential role in the process of RA‐induced cell differentiation. Therefore, we tested whether the PI3K/Akt pathway is involved in RA‐induced Hox gene expression in a F9 murine embryonic teratocarcinoma cells. To examine the effect of PI3K/Akt signaling on RA‐induced initiation of collinear expression of Hox genes, F9 cells were treated with RA in the presence or absence of PI3K inhibitor LY294002, and time‐course gene expression profiles for all 39 Hox genes located in four different clusters—Hoxa, Hoxb, Hoxc, and Hoxd—were analyzed. Collinear expression of Hoxa and ‐b cluster genes was initiated earlier than that of the ‐c and ‐d clusters upon RA treatment. When LY294002 was applied along with RA, collinear expression induced by RA was delayed, suggesting that the PI3K/Akt signaling pathway somehow regulates RA‐induced collinear expression of Hox genes in F9 cells. The initiation of Hox collinear expression by RA and the delayed expression following LY294002 in F9 cells would provide a good model system to decipher the yet to be answered de novo collinear expression of Hox genes during gastrulation, which make the gastrulating cells to remember their positional address along the AP body axis in the developing embryo.     

Expression cloning of Xenopus Os4, an evolutionarily conserved gene, which induces mesoderm and dorsal axis.

Multiple factors, including members of the FGF, TGF beta, and Wnt family of proteins, are important mediators in the regulation of dorsal-ventral pattern formation during vertebrate development. By using an expression cloning approach to identify novel factors that could regulate dorsal-ventral patterning in the Xenopus embryo, we isolated the Xenopus homologue of the human Os4 gene by virtue of its ability to induce a secondary dorsal axis. While Os4 homologues have been identified in a variety of species, and human Os4 is overexpressed in human tumors, the biological function of Os4 is unknown. To explore the mechanism by which Xenopus Os4 (XOs4) induces a secondary dorsal axis, we used Xenopus explant and whole-embryo assays. The secondary axis induced by XOs4 is distinct from that induced by activation of Wnt or FGF pathways but similar to that induced by inhibition of BMP signaling or activation of an Activin pathway. However, XOs4 did not inhibit BMP signaling in dissociated animal cap explants, indicating that XOs4 does not inhibit BMP signaling. Similar to activation of an Activin-like pathway, expression of XOs4 induces molecular markers for mesoderm in animal cap explants, although expression of gastrula-stage mesodermal markers was very weak and substantially delayed. Yet, XOs4 does not require activity of the Activin signal-transduction pathway for mesoderm induction as dominant-negative components of the Activin/Nodal/Vg1 pathway did not prevent XOs4-mediated induction of mesodermal derivatives. Finally, like Activin/Nodal/Vg1 pathways, XOs4 requires FGF signaling for expression of mesoderm markers. Results presented in this study demonstrate that XOs4 can induce mesoderm and dorsalize ventral mesoderm resulting in ectopic dorsal axis formation, suggesting a role for this large evolutionarily conserved gene family in early development.     

Activated G alpha q inhibits p110 alpha phosphatidylinositol 3-kinase and Akt

Some Gq-coupled receptors have been shown to antagonize growth factor activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. We used a constitutively active Galphaq(Q209L) mutant to explore the effects of Galphaq activation on signaling through the PI3K/Akt pathway. Transient expression of Galphaq(Q209L) in Rat-1 fibroblasts inhibited Akt activation induced by platelet-derived growth factor or insulin treatment. Expression of Galphaq(Q209L) also attenuated Akt activation promoted by coexpression of constitutively active PI3K in human embryonic kidney 293 cells. Galphaq(Q209L) had no effect on the activity of an Akt mutant in which the two regulatory phosphorylation sites were changed to acidic amino acids. Inducible expression of Galphaq(Q209L) in a stably transfected 293 cell line caused a decrease in PI3K activity in p110alpha (but not p110beta) immunoprecipitates. Receptor activation of Galphaq also selectively inhibited PI3K activity in p110alpha immunoprecipitates. Active Galphaq still inhibited PI3K/Akt in cells pretreated with the phospholipase C inhibitor U73122. Finally, Galphaq(Q209L) co-immunoprecipitated with the p110alpha-p85alpha PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated Galphaq(Q209L) with purified recombinant p110alpha-p85alpha in vitro led to a decrease in PI3K activity. These results suggest that agonist binding to Gq-coupled receptors blocks Akt activation via the release of active Galphaq subunits that inhibit PI3K. The inhibitory mechanism seems to be independent of phospholipase C activation and might involve an inhibitory interaction between Galphaq and p110alpha PI3K.     

Wnt3a regulates proliferation,apoptosis and function of pancreatic NIT‐1 beta cells via activation of IRS2/PI3K signaling

Wnt‐signaling pathway is implicated in pancreatic development and functional regulation of mature beta‐cells. Wnt3a/Wnt pathway activation expands islet cell mass in vitro by increasing proliferation and decreasing apoptosis of beta‐cells, thereby enhancing its function. However, the signaling pathways that mediate these effects remain unknown. By using a clonal beta‐cell line (NIT‐1), we examined the role of IRS2/PI3K in the mediation of Wnt3a‐stimulated beta‐cell growth. Real‐time PCR and Western blot were employed to investigate the activity of Wnt/β‐catenin and IRS2/PI3K signaling. Proliferation of NIT‐1 cells was assessed by BrdU incorporation, and apoptosis was quantitatively determined by TUNEL and flow cytometry (FCM). Dkk1, an inhibitor of Wnt signaling, and wortmannin, an inhibitor of PI3K, were also used. Results showed that Wnt3a rapidly activated Wnt/β‐catenin signaling, promoted IRS2 expression and Akt phosphorylation in NIT‐1 cells. These effects were completely abrogated by Dkk1 or partially eliminated by wortmannin. Wnt3a also promoted NIT‐1 cell proliferation, inhibited cytokine‐induced beta‐cell apoptosis, and increased insulin secretion. Both of these effects were also eliminated by Dkk1 or wortmannin. Our results demonstrated that Wnt3a regulates proliferation, apoptosis and enhances function of pancreatic NIT‐1 beta cells via activation of Wnt/β‐catenin signaling, involving crosstalk with IRS2/PI3K signaling, with the effect of Wnt signaling on beta‐cells also being IRS2/PI3K/AKT dependent. J. Cell. Biochem. 114: 1488–1497, 2013. © 2013 Wiley Periodicals, Inc.     

Galphaq negatively regulates the Wnt-beta-catenin pathway and dorsal embryonic Xenopus laevis development

The non-canonical Wnt/Ca2+ signaling pathway has been implicated in the regulation of axis formation and gastrulation movements during early Xenopus laevis embryo development, by antagonizing the canonical Wnt/beta-catenin dorsalizing pathway and specifying ventral cell fate. However, the molecular mechanisms involved in this antagonist crosstalk are not known. Since Galphaq is the main regulator of Ca2+ signaling in vertebrates and from this perspective probably involved in the events elicited by the non-canonical Wnt/Ca2+ pathway, we decided to study the effect of wild-type Xenopus Gq (xGalphaq) in dorso-ventral axis embryo patterning. Overexpression of xGalphaq or its endogenous activation at the dorsal animal region of Xenopus embryo both induced a strong ventralized phenotype and inhibited the expression of dorsal-specific mesoderm markers goosecoid and chordin. Dorsal expression of an xGalphaq dominant-negative mutant reverted the xGalphaq-induced ventralized phenotype. Finally, we observed that the Wnt8-induced secondary axis formation is reverted by endogenous xGalphaq activation, indicating that it is negatively regulating the Wnt/beta-catenin pathway.     

Biological functions of cytokeratin 18 in cancer

The structural proteins cytokeratin 18 (CK18) and its coexpressed complementary partner CK8 are expressed in a variety of adult epithelial organs and may play a role in carcinogenesis. In this study, we focused on the biological functions of CK18, which is thought to modulate intracellular signaling and operates in conjunction with various related proteins. CK18 may affect carcinogenesis through several signaling pathways, including the phosphoinositide 3-kinase (PI3K)/Akt, Wnt, and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathways. CK18 acts as an identical target of Akt in the PI3K/Akt pathway and of ERK1/2 in the ERK MAPK pathway, and regulation of CK18 by Wnt is involved in Akt activation. Finally, we discuss the importance of gaining a more complete understanding of the expression of CK18 during carcinogenesis, and suggest potential clinical applications of that understanding.     

Focal adhesion kinase protein regulates Wnt3a gene expression to control cell fate specification in the developing neural plate

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase protein localized to regions called focal adhesions, which are contact points between cells and the extracellular matrix. FAK protein acts as a scaffold to transfer adhesion-dependent and growth factor signals into the cell. Increased FAK expression is linked to aggressive metastatic and invasive tumors. However, little is known about its normal embryonic function. FAK protein knockdown during early Xenopus laevis development anteriorizes the embryo. Morphant embryos express increased levels of anterior neural markers, with reciprocally reduced posterior neural marker expression. Posterior neural plate folding and convergence-extension is also inhibited. This anteriorized phenotype resembles that of embryos knocked down zygotically for canonical Wnt signaling. FAK and Wnt3a genes are both expressed in the neural plate, and Wnt3a expression is FAK dependent. Ectopic Wnt expression rescues this FAK morphant anteriorized phenotype. Wnt3a thus acts downstream of FAK to balance anterior-posterior cell fate specification in the developing neural plate. Wnt3a gene expression is also FAK dependent in human breast cancer cells, suggesting that this FAK-Wnt linkage is highly conserved. This unique observation connects the FAK- and Wnt-signaling pathways, both of which act to promote cancer when aberrantly activated in mammalian cells.     

Identification of transmembrane protein 88 (TMEM88) as a dishevelled-binding protein

Wnt signaling pathways are involved in embryonic development and adult tissue maintenance and have been implicated in tumorigenesis. Dishevelled (Dvl/Dsh) protein is one of key components in Wnt signaling and plays essential roles in regulating these pathways through protein-protein interactions. Identifying and characterizing Dvl-binding proteins are key steps toward understanding biological functions. Given that the tripeptide VWV (Val-Trp-Val) binds to the PDZ domain of Dvl, we searched publically available databases to identify proteins containing the VWV motif at the C terminus that could be novel Dvl-binding partners. On the basis of the cellular localization and expression patterns of the candidates, we selected for further study the TMEM88 (target protein transmembrane 88), a two-transmembrane-type protein. The interaction between the PDZ domain of Dvl and the C-terminal tail of TMEM88 was confirmed by using NMR and fluorescence spectroscopy. Furthermore, in HEK293 cells, TMEM88 attenuated the Wnt/β-catenin signaling induced by Wnt-1 ligand in a dose-dependent manner, and TMEM88 knockdown by RNAi increased Wnt activity. In Xenopus, TMEM88 protein is sublocalized at the cell membrane and inhibits Wnt signaling induced by Xdsh but not β-catenin. In addition, TMEM88 protein inhibits the formation of a secondary axis normally induced by Xdsh. The findings suggest that TMEM88 plays a role in regulating Wnt signaling. Indeed, analysis of microarray data revealed that the expression of the Tmem88 gene was strongly correlated with that of Wnt signaling-related genes in embryonic mouse intestines. Together, we propose that TMEM88 associates with Dvl proteins and regulates Wnt signaling in a context-dependent manner.