Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts

ABSTRACTLarval tracheae of Drosophila harbor progenitors of the adult tracheal system (tracheoblasts). Thoracic tracheoblasts are arrested in the G2 phase of the cell cycle in an ATR (mei-41)-Checkpoint Kinase1 (grapes, Chk1) dependent manner prior to mitotic re-entry. Here we investigate developmental regulation of Chk1 activation. We report that Wnt signaling is high in tracheoblasts and is necessary for high levels of activated (phosphorylated) Chk1. We find that canonical Wnt signaling facilitates this by transcriptional upregulation of Chk1 expression in cells that have ATR kinase activity. Wnt signalling is dependent on four Wnts (Wg, Wnt5, 6,10) that are expressed at high levels in arrested tracheoblasts and downregulated at mitotic re-entry. Interestingly, none of the Wnts are dispensable and act synergistically to induce Chk1. Finally, we show that downregulation of Wnt signalling and Chk1 expression leads to mitotic re-entry and the concomitant upregulation of Dpp signalling, driving tracheoblast proliferation.

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Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts

eLife ◽

10.7554/elife.57056 ◽

2020 ◽

Vol 9 ◽

Author(s):

Amrutha Kizhedathu ◽

Rose Sebastian Kunnappallil ◽

Archit V Bagul ◽

Puja Verma ◽

Arjun Guha

Keyword(s):

Cell Cycle ◽

Wnt Signaling ◽

Kinase Activity ◽

Developmental Regulation ◽

Dependent Manner ◽

G2 Arrest ◽

Tracheal System ◽

G2 Phase ◽

Canonical Wnt ◽

Atr Kinase

Larval tracheae of Drosophila harbour progenitors of the adult tracheal system (tracheoblasts). Thoracic tracheoblasts are arrested in the G2 phase of the cell cycle in an ATR (mei-41)-Checkpoint Kinase1 (grapes, Chk1) dependent manner prior to mitotic re-entry. Here we investigate developmental regulation of Chk1 activation. We report that Wnt signaling is high in tracheoblasts and this is necessary for high levels of activated (phosphorylated) Chk1. We find that canonical Wnt signaling facilitates this by transcriptional upregulation of Chk1 expression in cells that have ATR kinase activity. Wnt signaling is dependent on four Wnts (Wg, Wnt5, 6,10) that are expressed at high levels in arrested tracheoblasts and are downregulated at mitotic re-entry. Interestingly, none of the Wnts are dispensable and act synergistically to induce Chk1. Finally, we show that downregulation of Wnt signaling and Chk1 expression leads to mitotic re-entry and the concomitant upregulation of Dpp signaling, driving tracheoblast proliferation.

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An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903418116 ◽

2019 ◽

Vol 116 (27) ◽

pp. 13374-13383 ◽

Cited By ~ 26

Author(s):

Tatiana N. Moiseeva ◽

Yandong Yin ◽

Michael J. Calderon ◽

Chenao Qian ◽

Sandra Schamus-Haynes ◽

...

Keyword(s):

Dna Replication ◽

Kinase Activity ◽

Kinase Inhibitors ◽

S Phase ◽

Dna Lesions ◽

Dependent Manner ◽

Kinase Signaling ◽

Origin Firing ◽

Atr Kinase ◽

Chk1 Kinase

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

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Relaxin reverses maladaptive remodeling of the aged heart through Wnt-signaling

Scientific Reports ◽

10.1038/s41598-019-53867-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Brian Martin ◽

Beth Gabris ◽

Amr F. Barakat ◽

Brian L. Henry ◽

Marianna Giannini ◽

...

Keyword(s):

Wnt Signaling ◽

Healthy Aging ◽

Ventricular Myocytes ◽

Therapeutic Potential ◽

Electrical Remodeling ◽

Dependent Manner ◽

Aged Rats ◽

Canonical Wnt ◽

Dickkopf 1 ◽

Fundamental Mechanism

AbstractHealthy aging results in cardiac structural and electrical remodeling that increases susceptibility to cardiovascular diseases. Relaxin, an insulin-like hormone, suppresses atrial fibrillation, inflammation and fibrosis in aged rats but the mechanisms-of-action are unknown. Here we show that relaxin treatment of aged rats reverses pathological electrical remodeling (increasing Nav1.5 expression and localization of Connexin43 to intercalated disks) by activating canonical Wnt signaling. In isolated adult ventricular myocytes, relaxin upregulated Nav1.5 (EC50 = 1.3 nM) by a mechanism inhibited by the addition of Dickkopf-1. Furthermore, relaxin increased the levels of connexin43, Wnt1, and cytosolic and nuclear β-catenin. Treatment with Wnt1 or CHIR-99021 (a GSK3β inhibitor) mimicked the relaxin effects. In isolated fibroblasts, relaxin blocked TGFβ-induced collagen elevation in a Wnt dependent manner. These findings demonstrate a close interplay between relaxin and Wnt-signaling resulting in myocardial remodeling and reveals a fundamental mechanism of great therapeutic potential.

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The canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2005.06.041 ◽

2005 ◽

Vol 333 (4) ◽

pp. 1300-1308 ◽

Cited By ~ 104

Author(s):

Fumiko Yano ◽

Fumitaka Kugimiya ◽

Shinsuke Ohba ◽

Toshiyuki Ikeda ◽

Hirotaka Chikuda ◽

...

Keyword(s):

Wnt Signaling ◽

Signaling Pathway ◽

Wnt Signaling Pathway ◽

Chondrocyte Differentiation ◽

Dependent Manner ◽

Canonical Wnt Signaling ◽

Canonical Wnt

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Analysis of Endogenous LRP6 Function Reveals a Novel Feedback Mechanism by Which Wnt Negatively Regulates Its Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.00773-07 ◽

2007 ◽

Vol 27 (20) ◽

pp. 7291-7301 ◽

Cited By ~ 51

Author(s):

Zahid Khan ◽

Sapna Vijayakumar ◽

Teresa Villanueva de la Torre ◽

Sabrina Rotolo ◽

Anna Bafico

Keyword(s):

Wnt Signaling ◽

Wnt Pathway ◽

Mrna Level ◽

Cellular Membrane ◽

Feedback Mechanism ◽

Bone Diseases ◽

Dependent Manner ◽

Direct Role ◽

Initial Stimulus ◽

Canonical Wnt

ABSTRACT The canonical Wnt pathway plays a crucial role in embryonic development, and its deregulation is involved in human diseases. The LRP6 single-span transmembrane coreceptor is essential for transmission of canonical Wnt signaling. However, due to the lack of immunological reagents, our understanding of LRP6 structure and function has relied on studies involving its overexpression, and regulation of the endogenous receptor by the Wnt ligand has remained unexplored. Using a highly sensitive and specific antibody to LRP6, we demonstrate that the endogenous receptor is modified by N-glycosylation and is phosphorylated in response to Wnt stimulation in a sustained yet ligand-dependent manner. Moreover, following triggering by Wnt, endogenous LRP6 is internalized and recycled back to the cellular membrane within hours of the initial stimulus. Finally, we have identified a novel feedback mechanism by which Wnt, acting through β-catenin, negatively regulates LRP6 at the mRNA level. Together, these findings contribute significantly to our understanding of LRP6 function and uncover a new level of regulation of Wnt signaling. In light of the direct role that the Wnt pathway plays in human bone diseases and malignancies, our findings may support the development of novel therapeutic approaches that target Wnt signaling through LRP6.

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Deregulation of the histone H3K9 di-methylation landscape suppresses canonical Wnt signaling in embryonal rhabdomyosarcoma

10.1101/2020.04.20.050120 ◽

2020 ◽

Author(s):

Ananya Pal ◽

Jia Yu Leung ◽

Gareth Chin Khye Ang ◽

Vinay Kumar Rao ◽

Luca Pignata ◽

...

Keyword(s):

Wnt Signaling ◽

Myogenic Differentiation ◽

Wnt Signaling Pathway ◽

Embryonal Rhabdomyosarcoma ◽

Dependent Manner ◽

Canonical Wnt Signaling ◽

Therapeutic Modalities ◽

Canonical Wnt

AbstractThe Wnt signaling pathway is down-regulated in embryonal rhabdomyosarcoma (ERMS) and contributes to the block of myogenic differentiation. Epigenetic mechanisms leading to its suppression are unknown and could pave the way towards novel therapeutic modalities. In this study, we demonstrate that the H3K9 lysine methyltransferase G9a suppresses canonical Wnt signaling by activating expression of the Wnt antagonist DKK1. Inhibition of G9a expression or activity reduced DKK1 expression and elevated canonical Wnt signaling resulting in myogenic differentiation in vitro and in vivo. Mechanistically, G9a impacted Sp1 and p300 enrichment at the DKK1 promoter in a methylation-dependent manner. The reduced tumor growth upon G9a deficiency was reversed by recombinant DKK1 or LGK974, which also inhibits Wnt signaling. Consistently, among thirteen drugs targeting chromatin modifiers, G9a inhibitors were highly effective in reducing ERMS cell viability. Together, our study demonstrates that ERMS cells are vulnerable to G9a inhibitors and suggest that targeting the G9a-DKK1-β-catenin node holds promise for differentiation therapy.

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Modulation of the canonical Wnt activity by androgen signaling in prostate epithelial basal stem cells

10.1101/2020.01.10.902270 ◽

2020 ◽

Author(s):

Yueli Liu ◽

Jiawen Wang ◽

Corrigan Horton ◽

Sol Katzman ◽

Tao Cai ◽

...

Keyword(s):

Stem Cell ◽

Wnt Signaling ◽

Target Genes ◽

Critical Role ◽

Gene Set Enrichment Analysis ◽

Dependent Manner ◽

Basal Cells ◽

Canonical Wnt Signaling ◽

Cell Functions ◽

Canonical Wnt

AbstractBoth the canonical Wnt signaling and androgen signaling are important factors regulating prostate organogenesis. How these two pathways crosstalk to regulate prostate stem cell functions remain unclear. Here, we show that while canonical Wnt activity is required for prostate basal stem cell multipotency in vivo, ectopic Wnt activity does not promote basal-to-luminal cell differentiation. We provide evidence that androgen signaling may keep Wnt activity in check. In prostate organoid culture from basal cells, dihydrotestosterone (DHT) antagonizes R-spondin-stimulated organoid growth in a concentration-dependent manner. Molecular analyses of organoids under different treatment conditions showed that androgen signaling down-regulated the expressions of a Wnt reporter as well as many Wnt target genes. Pathway analysis and gene set enrichment analysis of organoid RNA-seq data also revealed the canonical Wnt signaling as a key pathway distinguishing organoids treated with or without DHT. Notably, DHT treatment enhanced AR and β–catenin binding in the nuclei of prostate organoids, providing possible mechanistic clues. Our results reveal a critical role of AR signaling in modulating canonical Wnt activity in prostate basal cells to regulate their multipotency.

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Wnt-5a Promotes Neural Development and Differentiation by Regulating CDK5 via Ca2+/Calpain Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000495932 ◽

2018 ◽

Vol 51 (6) ◽

pp. 2604-2615 ◽

Cited By ~ 5

Author(s):

Yang Shu ◽

Min Xiang ◽

Pei Zhang ◽

Guangjian Qi ◽

Feng He ◽

...

Keyword(s):

Cell Differentiation ◽

Wnt Signaling ◽

Neural Development ◽

Kinase Activity ◽

Neuronal Development ◽

Cell Model ◽

Wnt Signaling Pathway ◽

Specific Chemical ◽

Development And Differentiation ◽

Canonical Wnt

Background/Aims: The Wnt signaling pathway has essential functions in the central nervous system, where it regulates the major physiological functions of neurons, including development, differentiation, and plasticity. Wnt signaling controls these cellular events; however, how Wnt pathways integrate into a coherent developmental program remains unclear. Methods: The expression and secretion of different WNT ligands (Wnt-1, Wnt-3a, Wnt-4, Wnt-5a, Wnt-11), and the levels and activities of cyclin-dependent kinases (CDK2, CDK4, CDK6/cyclin D, cyclin E) or CDK5 (CDK5/p35 and p25) were measured in Rat cortex at different embryonic stages, and in RA/BDNF-induced differentiated SH-SY5Y cell model, by Quantitative real-time PCR (qPCR), western blotting, ELISA, and in vitro CDK5 kinase assays. MAP2-BrdU double staining was used to assess cell differentiation and cell cycle exit in an RA/BDNF-induced differentiated SH-SY5Y cell model. The effects of CDK5 and Ca2+/calpain signaling were assessed using specific chemical inhibitors. Results: We found that Wnt-1 was unchanged and Wnt-3a was attenuated, whereas Wnt-4, Wnt-5a, and Wnt-11 were markedly up-regulated, during the development of neurons and differentiated SH-SY5Y cells. Simultaneously, the activity of CDK5 was elevated. Furthermore, we describe crosstalk between non-canonical Wnt signaling and CDK5 in the development of neurons and differentiated SH-SY5Y cells. Wnt-5a, a non-canonical Wnt ligand, regulated CDK5 via Ca2+/calpain signaling in both neuronal development and differentiation. Inhibition of Wnt-5a diminished CDK5 kinase activity via the Ca2+/calpain pathway, thereby attenuating RA-BDNF induced SH-SY5Y cell differentiation. Conclusion: Wnt-5a signaling is a significant regulator of neuronal development and differentiation and upregulates CDK5 kinase activity via Ca2+/calpain signaling.

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Chlorogenic Acids Inhibit Adipogenesis: Implications of Wnt/β-Catenin Signaling Pathway

International Journal of Endocrinology ◽

10.1155/2021/2215274 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Mengting Liu ◽

Jian Qin ◽

Jing Cong ◽

Yubin Yang

Keyword(s):

Wnt Signaling ◽

Signaling Pathway ◽

Fatty Acid Synthase ◽

Adipocyte Differentiation ◽

Glycogen Synthase ◽

Dependent Manner ◽

Canonical Wnt Signaling ◽

Ppar Γ ◽

Gsk 3Β ◽

Canonical Wnt

In our previous in vitro study, we found that chlorogenic acid (CGA) inhibited adipocyte differentiation and triglyceride (TG) accumulation, but the underlying mechanism is still unclear. Accumulative genetic evidence supports that canonical Wnt signaling is a key modulator on adipogenesis. Methods. In this study, 3T3-L1 cells were induced adipogenic differentiation and then treated with CGA. We investigate the effect of CGA in inhibiting adipogenesis and evaluate its role in modulating Wnt10b (wingless integration1 10b), β-catenin, glycogen synthase kinase-3β (GSK-3β), and peroxisome proliferator-activated receptor γ (PPAR-γ) involved in the Wnt (wingless integration1)/β-catenin signaling pathway. Results. The result showed that after CGA treatment, lipid accumulation and TG level decreased significantly in 3T3-L1 cells, indicating that CGA could inhibit adipogenesis. In addition, CGA repressed the induction of adipocyte differentiation biomarkers as PPAR-γ, adipocyte protein 2 (aP2), fatty acid synthase (FAS), and lipoprotein lipase (LPL), and the secretion of GSK-3β in a dose-dependent manner upregulated the expression of β-catenin and Wnt10b both in gene and protein levels. Moreover, CGA induced phosphorylation of GSK-3β and promoted the accumulation of free cytosolic β-catenin in 3T3-L1 adipocytes. Conclusion. Overall, these findings gave us the implications that CGA inhibits adipogenesis via the canonical Wnt signaling pathway.

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Spatiotemporal regulations of Wee1 at the G2/M transition

Molecular Biology of the Cell ◽

10.1091/mbc.e10-07-0644 ◽

2011 ◽

Vol 22 (5) ◽

pp. 555-569 ◽

Cited By ~ 20

Author(s):

Hirohisa Masuda ◽

Chii Shyang Fong ◽

Chizuru Ohtsuki ◽

Tokuko Haraguchi ◽

Yasushi Hiraoka

Keyword(s):

Feedback Loop ◽

Kinase Activity ◽

Spindle Pole Body ◽

Spindle Assembly ◽

Spindle Pole ◽

Cyclin B ◽

Dependent Manner ◽

Mitotic Entry ◽

G2 Phase ◽

Cdc2 Activity

Wee1 is a protein kinase that negatively regulates mitotic entry in G2 phase by suppressing cyclin B–Cdc2 activity, but its spatiotemporal regulations remain to be elucidated. We observe the dynamic behavior of Wee1 in Schizosaccharomyces pombe cells and manipulate its localization and kinase activity to study its function. At late G2, nuclear Wee1 efficiently suppresses cyclin B–Cdc2 around the spindle pole body (SPB). During the G2/M transition when cyclin B–Cdc2 is highly enriched at the SPB, Wee1 temporally accumulates at the nuclear face of the SPB in a cyclin B–Cdc2-dependent manner and locally suppresses both cyclin B–Cdc2 activity and spindle assembly to counteract a Polo kinase–dependent positive feedback loop. Then Wee1 disappears from the SPB during spindle assembly. We propose that regulation of Wee1 localization around the SPB during the G2/M transition is important for proper mitotic entry and progression.

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