scholarly journals Calreticulin regulates a switch between osteoblast and chondrocyte lineages derived from murine embryonic stem cells

2020 ◽  
Vol 295 (20) ◽  
pp. 6861-6875 ◽  
Author(s):  
Carlos Pilquil ◽  
Zahra Alvandi ◽  
Michal Opas
Keyword(s):  
Endoplasmic Reticulum ◽  
Glycogen Synthase ◽  
Wnt Signaling Pathway ◽  
Embryonic Stem ◽  
Activated T Cells ◽  
Endoplasmic Reticulum Calcium ◽  
Chemically Induced ◽  
Enhanced Expression ◽  
Intranuclear Transport ◽  
Canonical Wnt

Calreticulin is a highly conserved, ubiquitous Ca2+-buffering protein in the endoplasmic reticulum that controls transcriptional activity of various developmental programs and also of embryonic stem cell (ESC) differentiation. Calreticulin activates calcineurin, which dephosphorylates and induces the nuclear import of the osteogenic transcription regulator nuclear factor of activated T cells 1 (NFATC1). We investigated whether calreticulin controls a switch between osteogenesis and chondrogenesis in mouse ESCs through NFATC1. We found that in the absence of calreticulin, intranuclear transport of NFATC1 is blocked and that differentiation switches from osteogenic to chondrogenic, a process that could be mimicked by chemical inhibition of NFAT translocation. Glycogen synthase kinase 3β (GSK3β) deactivation and nuclear localization of β-catenin critical to osteogenesis were abrogated by calreticulin deficiency or NFAT blockade. Chemically induced GSK3β inhibition bypassed the calreticulin/calcineurin axis and increased osteoblast output from both control and calreticulin-deficient ESCs, while suppressing chondrogenesis. Calreticulin-deficient ESCs or cells treated with an NFAT blocker had enhanced expression of dickkopf WNT-signaling pathway inhibitor 1 (Dkk1), a canonical Wnt pathway antagonist that blocks GSK3β deactivation. The addition of recombinant mDKK1 switched osteogenic ESC differentiation toward chondrogenic differentiation. The results of our study indicate a role for endoplasmic reticulum calcium signaling via calreticulin in the differentiation of ESCs to closely associated osteoblast or chondrocyte lineages.

scholarly journals A divergent canonical WNT-signaling pathway regulates microtubule dynamics

2004 ◽  
Vol 164 (2) ◽  
pp. 243-253 ◽  
Author(s):  
Lorenza Ciani ◽  
Olga Krylova ◽  
Matthew J. Smalley ◽  
Trevor C. Dale ◽  
Patricia C. Salinas
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Glycogen Synthase ◽  
Wnt Signaling Pathway ◽  
Negative Regulator ◽  
Canonical Wnt Signaling ◽  
Microtubule Stability ◽  
Gsk 3Β ◽  
Canonical Wnt

Dishevelled (DVL) is associated with axonal microtubules and regulates microtubule stability through the inhibition of the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β). In the canonical WNT pathway, the negative regulator Axin forms a complex with β-catenin and GSK-3β, resulting in β-catenin degradation. Inhibition of GSK-3β by DVL increases β-catenin stability and TCF transcriptional activation. Here, we show that Axin associates with microtubules and unexpectedly stabilizes microtubules through DVL. In turn, DVL stabilizes microtubules by inhibiting GSK-3β through a transcription- and β-catenin–independent pathway. More importantly, axonal microtubules are stabilized after DVL localizes to axons. Increased microtubule stability is correlated with a decrease in GSK-3β–mediated phosphorylation of MAP-1B. We propose a model in which Axin, through DVL, stabilizes microtubules by inhibiting a pool of GSK-3β, resulting in local changes in the phosphorylation of cellular targets. Our data indicate a bifurcation in the so-called canonical WNT-signaling pathway to regulate microtubule stability.

scholarly journals Neural differentiation is increased by GSK-3β inhibition and decreased by tankyrase inhibition in human neural precursor cells

2019 ◽  
Author(s):  
Michael Telias ◽  
Dalit Ben-Yosef
Keyword(s):  
Cell Biology ◽  
Neural Differentiation ◽  
Glycogen Synthase ◽  
Embryonic Stem ◽  
Precursor Cells ◽  
Neural Precursor Cells ◽  
Neural Precursor ◽  
Therapeutic Drugs ◽  
Gsk 3Β ◽  
Canonical Wnt

AbstractGlycogen synthase kinase-3β (GSK-3β) and tankyrase-1/2 (TANK) are two enzymes known to play multiple roles in cell biology, including regulation of proliferation, differentiation and metabolism. Both of them act on the canonical Wnt/β-Catenin pathway, but are also involved in many other independent intracellular mechanisms. More importantly, GSK-3β and TANK have been shown to play crucial roles in different diseases, including cancer and neurological disorders. The GSK-3β-inhibitor ‘CHIR’ and the TANK-inhibitor ‘XAV’ are two pyrimidine molecules, holding high potential as possible therapeutic drugs. However, their effect on neural tissue is poorly understood. In this study, we tested the effects of CHIR and XAV on human neural precursor cells (hNPCs) derived from human embryonic stem cells. We found that CHIR-mediated inhibition of GSK-3β promotes neural differentiation. In contrast, XAV-mediated inhibition of TANK leads to de-differentiation. These results highlight the relative importance of these two enzymes in determining the neurodevelopmental status of hNPCs. Furthermore, they shed light on the roles of Wnt signaling during early human neurogenesis.

164 POTENTIAL REGULATION OF PERI-IMPLANTATION OVINE CONCEPTUS GROWTH AND DIFFERENTIATION BY THE WNT SIGNALING SYSTEM

2007 ◽  
Vol 19 (1) ◽  
pp. 199
Author(s):  
K. Hayashi ◽  
R. C. Burghardt ◽  
F. W. Bazer ◽  
T. E. Spencer
Keyword(s):  
Wnt Signaling ◽  
Cell Fate ◽  
Target Genes ◽  
Glycogen Synthase ◽  
Wnt Signaling Pathway ◽  
Density Lipoprotein ◽  
Cell Types ◽  
Negative Regulator ◽  
Beta Catenin ◽  
Canonical Wnt

In mice WNT signaling regulates cell fate, differentiation, and growth in the conceptus (embryo and associated extra-embryonic membranes), as well as implantation. We studied various components of the WNT signaling pathway in the ovine uterus during the estrous cycle (C) and pregnancy (P) and in the peri-implantation conceptus. Expression of WNT2, WNT2B, and WNT4 mRNAs was very low in endometria of C and P ewes from Days 10 to 16 and in conceptus trophectoderm (Tr). WNT5A/5B mRNAs were abundant in the stratum compactum stroma, whereas WNT11 mRNA was detected in endometrial epithelia of C and P ewes, but not in conceptus Tr. WNT7A mRNA was localized specifically to luminal (LE) and superficial glandular (sGE) epithelia of Day 10 C and P ewes, was undetectable by Day 12, and then increased up to Day 16 and was maximum on Day 20 only in P ewes. Frizzled receptor (FZD6/8) mRNAs were detected primarily in conceptus Tr and uterine LE and GE, whereas the co-receptor LRP5/6 (low density lipoprotein receptor-related protein) mRNAs were expressed in all uterine cell types and conceptus Tr. Dickkopf (DKK1), a negative regulator of WNT signaling, was detected in stratum compactum stroma after Day 14 in P ewes. CTNNB1 (beta-catenin), a key mediator of canonical WNT signaling, and GSK3B (glycogen synthase kinase-3 beta) and CHD1 (E-cadherin) mRNAs were abundant in endometrial epithelia and in conceptus Tr. Immunoreactive CTNNB1 protein was abundant in LE and GE, and present at lower levels in stroma and myometrium in uteri from C and P ewes. In the conceptus Tr, immunoreactive CTNNB1 protein was abundant in nuclei of the mononuclear and binuclear cells (BNC), as well as in cell adherens junctions. Nuclear CTNNB1 interacts with transcription factors, most notably LEF1/TCF7 (lymphoid enhancer-binding factor 1/transcription factor 7), to regulate gene transcription. LEF1 mRNA was detected in LE and sGE, whereas nuclear TCF7L2 protein was particularly abundant in trophoblast giant BNC. WNT/beta-catenin/TCF7 target genes were also studied. MSX2 mRNA was abundant in conceptus Tr, and MYC mRNA was abundant in BNC of conceptus Tr and endometrial epithelia. Next, ovine Tr (oTr) cells and endometrial stromal (oST) cells were used for mechanistic studies that revealed that transfection of mouse WNT7A stimulated a LEF/TCF-responsive reporter (TOPFLASH), and co-transfection of either dnTCF or SFRP2 (a secreted FZD inhibitor) inhibited WNT7A effects. WNT7A stimulated expression of MSX2 and MYC in oTr cells, and this effect was inhibited by SFRP2. These results implicate the canonical WNT system as a regulator of peri-implantation conceptus growth and differentiation in sheep. This work was supported by NIH HD38274 and 5 P30 ES09106 funding.

scholarly journals Interaction among Gsk-3, Gbp, Axin, and APC in Xenopus Axis Specification

2000 ◽  
Vol 148 (4) ◽  
pp. 691-702 ◽  
Author(s):  
Gist H. Farr ◽  
Denise M. Ferkey ◽  
Cynthia Yost ◽  
Sarah B. Pierce ◽  
Carole Weaver ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Glycogen Synthase ◽  
Wnt Signaling Pathway ◽  
Early Embryo ◽  
Dominant Negative ◽  
Axis Formation ◽  
Downstream Effector ◽  
Axis Specification ◽  
Subsequent Degradation ◽  
Canonical Wnt

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is β-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal–ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and β-catenin form a complex that promotes the GSK-3–mediated phosphorylation and subsequent degradation of β-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates β-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3–mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic β-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in β-catenin levels and establishment of the dorsal axis.

scholarly journals Fas-associated factor 1 antagonizes Wnt signaling by promoting β-catenin degradation

2011 ◽  
Vol 22 (9) ◽  
pp. 1617-1624 ◽  
Author(s):  
Long Zhang ◽  
Fangfang Zhou ◽  
Theo van Laar ◽  
Juan Zhang ◽  
Hans van Dam ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathway ◽  
Glycogen Synthase ◽  
Ectopic Expression ◽  
Wnt Signaling Pathway ◽  
Negative Regulator ◽  
Associated Factor ◽  
Functional Studies ◽  
Cell Proliferation And Differentiation ◽  
Canonical Wnt

The canonical Wnt pathway plays an important role in the regulation of cell proliferation and differentiation. Activation of this signaling pathway causes disruption of the Axin/adenomatous polyposis coli/glycogen synthase kinase 3β complex, resulting in stabilization of β-catenin and its association with lymphoid enhancer factor/T-cell factor in the nucleus. Here, we identify Fas-associated factor 1 (FAF1) as a negative regulator of Wnt/β-catenin signaling. We found overexpression of FAF1 to strongly inhibit Wnt-induced transcriptional reporter activity and to counteract Wnt-induced β-catenin accumulation. Moreover, knockdown of FAF1 resulted in an increase in β-catenin levels and in activation of Wnt/β-catenin–induced transcription. FAF1 was found to interact with β-catenin upon inhibition of proteasome. Ectopic expression of FAF1 promoted β-catenin degradation by enhancing its polyubiquitination. Functional studies in C2C12 myoblasts and KS483 preosteoblastic cells showed that FAF1 depletion resulted in activation of endogenous Wnt-induced genes and enhanced osteoblast differentiation, whereas FAF1 overexpression had the opposite effect. These results identify FAF1 as a novel inhibitory factor of canonical Wnt signaling pathway.

scholarly journals Caprin-2 enhances canonical Wnt signaling through regulating LRP5/6 phosphorylation

2008 ◽  
Vol 182 (5) ◽  
pp. 865-872 ◽  
Author(s):  
Yu Ding ◽  
Ying Xi ◽  
Ting Chen ◽  
Ji-yong Wang ◽  
Dong-lei Tao ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Mammalian Cells ◽  
Target Genes ◽  
Glycogen Synthase ◽  
Signal Transmission ◽  
Low Density Lipoprotein ◽  
Wnt Signaling Pathway ◽  
Density Lipoprotein ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt

The low-density lipoprotein receptor–related proteins 5 and 6 (LRP5/6) are coreceptors for Frizzled and transmit signals from the plasma membrane to the cytosol. However, the mechanism for LRP5/6 signal transmission remains undefined. Here, we identify cytoplasmic activation/proliferation-associated protein 2 (Caprin-2) as a LRP5/6-binding protein. Our data show that Caprin-2 stabilizes cytosolic β-catenin and enhances lymphoid enhancer-binding factor 1/T cell factor–dependent reporter gene activity as well as the expression of Wnt target genes in mammalian cells. Morpholino-mediated knockdown of Caprin-2 in zebrafish embryos inhibits Wnt/β-catenin signaling and results in a dorsalized phenotype. Moreover, Caprin-2 facilitates LRP5/6 phosphorylation by glycogen synthase kinase 3, and thus enhances the interaction between Axin and LRP5/6. Therefore, Caprin-2 promotes activation of the canonical Wnt signaling pathway by regulating LRP5/6 phosphorylation.

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