12-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in biomedical studies. TPA has been proposed as a pluripotent cell differentiation factor, but results obtained have been inconsistent. In the present study we show that TPA can be applied as a cardiomyogenesis-promoting factor for the differentiation of mouse embryonic stem (mES) cells in vitro. The mechanism of TPA action is mediated by the induction of extracellular signal-regulated kinase (ERK) activity and the subsequent phosphorylation of GATA4 transcription factor. Interestingly, general mitogens (FGF, EGF, VEGF and serum) or canonical WNT signalling did not mimic the effect of TPA. Moreover, on the basis of our results, we postulate that a TPA-sensitive population of cardiac progenitor cells exists at a certain time point (after days 6-8 of the differentiation protocol) and that the proposed treatment can be used to increase the multiplication of ES cell-derived cardiomyocytes.

• MeSH
• Cell Differentiation drug effects   MeSH
• Embryonic Stem Cells cytology   drug effects   metabolism   MeSH
• Extracellular Signal-Regulated MAP Kinases metabolism   MeSH
• Phosphorylation MeSH
• Myocytes, Cardiac cytology   metabolism   MeSH
• Mice MeSH
• Protein Kinase C metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Tetradecanoylphorbol Acetate pharmacology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Extracellular Signal-Regulated MAP Kinases MeSH
• Protein Kinase C MeSH
• Tetradecanoylphorbol Acetate MeSH

Wnt signaling cascade has developed together with multicellularity to orchestrate the development and homeostasis of complex structures. Wnt pathway components - such as β-catenin, Dishevelled (DVL), Lrp6, and Axin-- are often dedicated proteins that emerged in evolution together with the Wnt signaling cascade and are believed to function primarily in the Wnt cascade. It is interesting to see that in recent literature many of these proteins are connected with cellular functions that are more ancient and not limited to multicellular organisms - such as cell cycle regulation, centrosome biology, or cell division. In this review, we summarize the recent literature describing this crosstalk. Specifically, we attempt to find the answers to the following questions: Is the response to Wnt ligands regulated by the cell cycle? Is the centrosome and/or cilium required to activate the Wnt pathway? How do Wnt pathway components regulate the centrosomal cycle and cilia formation and function? We critically review the evidence that describes how these connections are regulated and how they help to integrate cell-to-cell communication with the cell and the centrosomal cycle in order to achieve a fine-tuned, physiological response.

• Keywords
• Wnt, cell cycle, centrosome, cilium, crosstalk, planar cell polarity,
• MeSH
• Cell Cycle * MeSH
• Centrosome metabolism   MeSH
• Humans MeSH
• Cell Communication MeSH
• Cell Polarity MeSH
• Wnt Signaling Pathway * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.

• MeSH
• Acetophenones pharmacology   MeSH
• Extracellular Signal-Regulated MAP Kinases metabolism   MeSH
• Phosphatidylinositol 3-Kinases metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Mouse Embryonic Stem Cells drug effects   metabolism   MeSH
• Mice MeSH
• NADPH Oxidases genetics   metabolism   MeSH
• Onium Compounds pharmacology   MeSH
• Oxidative Stress drug effects   MeSH
• Cell Proliferation drug effects   MeSH
• Wnt Proteins metabolism   MeSH
• Proto-Oncogene Proteins c-akt metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Drug Synergism MeSH
• STAT3 Transcription Factor metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acetophenones MeSH
• acetovanillone MeSH Browser
• diphenyleneiodonium MeSH Browser
• Extracellular Signal-Regulated MAP Kinases MeSH
• Phosphatidylinositol 3-Kinases MeSH
• NADPH Oxidases MeSH
• Onium Compounds MeSH
• Wnt Proteins MeSH
• Proto-Oncogene Proteins c-akt MeSH
• Reactive Oxygen Species MeSH
• STAT3 Transcription Factor MeSH

The seven-transmembrane-spanning receptors of the FZD1-10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD2, FZD4, or FZD5, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD2/4/5 as measured by phosphorylation of LRP6 and β-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.

• Keywords
• 32D Cells, Disheveled, Frizzled, Functional Selectivity, LDL Receptor-related Protein 6, Myeloid Cell, Receptor, WNT Pathway, WNT Signaling, β-Catenin (B-catenin),
• MeSH
• beta Catenin metabolism   MeSH
• Cell Line MeSH
• Phosphorylation MeSH
• Frizzled Receptors metabolism   MeSH
• Protein Interaction Mapping MeSH
• Protein Interaction Maps * MeSH
• Mice MeSH
• Protein Isoforms metabolism   MeSH
• Wnt Proteins metabolism   MeSH
• Wnt Signaling Pathway * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• beta Catenin MeSH
• Frizzled Receptors MeSH
• Protein Isoforms MeSH
• Wnt Proteins MeSH

β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.

• Keywords
• Amer1/WTX/FAM123B, Dvl, Lrp6 Phosphorylation, Membrane Lipids, Phosphatidylinositol Kinase, Phosphatidylinositol Phosphate Kinase, Phosphatidylinositol Signaling, Wnt Signaling, β-Arrestin, β-Catenin,
• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Arrestins genetics   metabolism   MeSH
• beta-Arrestins MeSH
• Cell Membrane metabolism   MeSH
• Embryo, Mammalian cytology   MeSH
• Fibroblasts cytology   metabolism   MeSH
• Phosphatidylinositol 4,5-Diphosphate metabolism   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Phosphorylation MeSH
• Phosphotransferases (Alcohol Group Acceptor) genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Microscopy, Confocal MeSH
• Cells, Cultured MeSH
• Low Density Lipoprotein Receptor-Related Protein-6 genetics   metabolism   MeSH
• Humans MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Tumor Suppressor Proteins genetics   metabolism   MeSH
• Dishevelled Proteins MeSH
• Wnt3A Protein genetics   metabolism   MeSH
• RNA Interference MeSH
• Protein Binding MeSH
• Minor Histocompatibility Antigens MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-phosphatidylinositol-4-phosphate 5-kinase MeSH Browser
• Adaptor Proteins, Signal Transducing MeSH
• AMER1 protein, human MeSH Browser
• Arrestins MeSH
• beta-Arrestins MeSH
• Phosphatidylinositol 4,5-Diphosphate MeSH
• Phosphoproteins MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Low Density Lipoprotein Receptor-Related Protein-6 MeSH
• LRP6 protein, human MeSH Browser
• Tumor Suppressor Proteins MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Browser
• Dishevelled Proteins MeSH
• Wnt3A Protein MeSH
• Minor Histocompatibility Antigens MeSH