Human embryo polarization requires PLC signaling to mediate trophectoderm specification.

Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8-16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the eight-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos.

Differential requirements for MDM2 E3 activity during embryogenesis and in adult mice.

The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo, constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities.

Phosphodiesterase 7 Regulation in Cellular and Rodent Models of Parkinson's Disease.

Parkinson's disease is characterized by a loss of dopaminergic neurons in the ventral midbrain. This disease is diagnosed when around 50% of these neurons have already died; consequently, therapeutic treatments start too late. Therefore, an urgent need exists to find new targets involved in the onset and progression of the disease. Phosphodiesterase 7 (PDE7) is a key enzyme involved in the degradation of intracellular levels of cyclic adenosine 3', 5'-monophosphate in different cell types; however, little is known regarding its role in neurodegenerative diseases, and specifically in Parkinson's disease. We have previously shown that chemical as well as genetic inhibition of this enzyme results in neuroprotection and anti-inflammatory activity in different models of neurodegenerative disorders, including Parkinson's disease. Here, we have used in vitro and in vivo models of Parkinson's disease to study the regulation of PDE7 protein levels. Our results show that PDE7 is upregulated after an injury both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures and after lipopolysaccharide or 6-hidroxydopamine injection in the Substantia nigra pars compacta of adult mice. PDE7 increase takes place mainly in degenerating dopaminergic neurons and in microglia cells. This enhanced expression appears to be direct since 6-hydroxydopamine and lipopolysaccharide increase the expression of a 962-bp fragment of its promoter. Taking together, these results reveal an essential function for PDE7 in the pathways leading to neurodegeneration and inflammatory-mediated brain damage and suggest novel roles for PDE7 in neurodegenerative diseases, specifically in PD, opening the door for new therapeutic interventions.

Halogen Bonding Increases the Potency and Isozyme Selectivity of Protein Arginine Deiminase 1 Inhibitors.

Protein arginine deiminases (PADs) hydrolyze the side chain of arginine to form citrulline. Aberrant PAD activity is associated with rheumatoid arthritis, multiple sclerosis, lupus, and certain cancers. These pathologies established the PADs as therapeutic targets and multiple PAD inhibitors are known. Herein, we describe the first highly potent PAD1-selective inhibitors (1 and 19). Detailed structure-activity relationships indicate that their potency and selectivity is due to the formation of a halogen bond with PAD1. Importantly, these inhibitors inhibit histone H3 citrullination in HEK293TPAD1 cells and mouse zygotes with excellent potency. Based on this scaffold, we also developed a PAD1-selective activity-based probe that shows remarkable cellular efficacy and proteome selectivity. Based on their potency and selectivity we expect that 1 and 19 will be widely used chemical tools to understand PAD1 biology.

The Pol ß variant containing exon α is deficient in DNA polymerase but has full dRP lyase activity.

DNA polymerase (Pol) ß is a key enzyme in base excision repair (BER), an important repair system for maintaining genomic integrity. We previously reported the presence of a Pol ß transcript containing exon α (105-nucleotide) in normal and colon cancer cell lines. The transcript carried an insertion between exons VI and VII and was predicted to encode a ~42 kDa variant of the wild-type 39 kDa enzyme. However, little is known about the biochemical properties of the exon α-containing Pol ß (exon α Pol ß) variant. Here, we first obtained evidence indicating expression of the 42 kDa exon α Pol ß variant in mouse embryonic fibroblasts. The exon α Pol ß variant was then overexpressed in E. coli, purified, and characterized for its biochemical properties. Kinetic studies of exon α Pol ß revealed that it is deficient in DNA binding to gapped DNA, has strongly reduced polymerase activity and higher Km for dNTP during gap-filling. On the other hand, the 5'-dRP lyase activity of the exon α Pol ß variant is similar to that of wild-type Pol ß. These results indicate the exon α Pol ß variant is base excision repair deficient, but does conduct 5'-trimming of a dRP group at the gap margin. Understanding the biological implications of this Pol ß variant warrants further investigation.

Pathological mTOR mutations impact cortical development.

Several mosaic mutations of the mammalian/mechanistic target of rapamycin (mTOR) have recently been found in patients with cortical malformations, such as hemimegalencephaly (HME) and focal cortical dysplasia (FCD). Although all of them should activate mTOR signaling, comparisons of the impact of different mTOR mutations on brain development have been lacking. Also it remains unknown if any potential differences these mutations may have on cortical development are directly related to a degree of mTOR signaling increase. The present study assessed levels of mTORC1 pathway activity in cell lines and rat primary neurons overexpressing several mTOR mutants that were previously found in HME, FCD, cancer patients and in vitro mutagenesis screens. Next we introduced the mutants, enhancing mTORC1 signaling most potently, into developing mouse brains and assessed electroporated cell morphology and migratory phenotype using immunofluorescent staining. We observed the differential inhibition of neuronal progenitor cortical migration, which partly corresponded with a degree of mTORC1 signaling enhancement these mutants induced in cultured cells. The most potent quadruple mutant prevented most of the progenitors from entering the cortical plate. Cells that expressed less potent, single-point, mTOR mutants entered the cortical plate but failed to reach its upper layers and had enlarged soma. Our findings suggest a correlation between the potency of mTOR mutation to activate mTORC1 pathway and disruption of cortical migration.

Premature activation of Cdk1 leads to mitotic events in S phase and embryonic lethality.

Cell cycle regulation, especially faithful DNA replication and mitosis, are crucial to maintain genome stability. Cyclin-dependent kinase (CDK)/cyclin complexes drive most processes in cellular proliferation. In response to DNA damage, cell cycle surveillance mechanisms enable normal cells to arrest and undergo repair processes. Perturbations in genomic stability can lead to tumor development and suggest that cell cycle regulators could be effective targets in anticancer therapy. However, many clinical trials ended in failure due to off-target effects of the inhibitors used. Here, we investigate in vivo the importance of WEE1- and MYT1-dependent inhibitory phosphorylation of mammalian CDK1. We generated Cdk1AF knockin mice, in which two inhibitory phosphorylation sites are replaced by the non-phosphorylatable amino acids T14A/Y15F. We uncovered that monoallelic expression of CDK1AF is early embryonic lethal in mice and induces S phase arrest accompanied by γH2AX and DNA damage checkpoint activation in mouse embryonic fibroblasts (MEFs). The chromosomal fragmentation in Cdk1AF MEFs does not rely on CDK2 and is partly caused by premature activation of MUS81-SLX4 structure-specific endonuclease complexes, as well as untimely onset of chromosome condensation followed by nuclear lamina disassembly. We provide evidence that tumor development in liver expressing CDK1AF is inhibited. Interestingly, the regulatory mechanisms that impede cell proliferation in CDK1AF expressing cells differ partially from the actions of the WEE1 inhibitor, MK-1775, with p53 expression determining the sensitivity of cells to the drug response. Thus, our work highlights the importance of improved therapeutic strategies for patients with various cancer types and may explain why some patients respond better to WEE1 inhibitors.

The eggstraordinary story of how life begins.

More than 15 years have elapsed since the identification of phospholipase C ζ1 (PLCζ) from a genomic search for mouse testis/sperm-specific PLCs. This molecule was proposed to represent the sperm factor responsible for the initiation of calcium (Ca2+ ) oscillations required for egg activation and embryo development in mammals. Supporting evidence for this role emerged from studies documenting its expression in all mammals and other vertebrate species, the physiological Ca2+ rises induced by injection of its messenger RNA into mammalian and nonmammalian eggs, and the lack of expression in infertile males that fail intracytoplasmic sperm injection. In the last year, genetic animal models have added support to its role as the long sought-after sperm factor. In this review, we highlight the findings that demonstrated the role of Ca2+ as the universal signal of egg activation and the experimental buildup that culminated with the identification of PLCζ as the soluble sperm factor. We also discuss the structural-functional properties that make PLCζ especially suited to evoke oscillations in eggs. Lastly, we examine unresolved aspects of the function and regulation of PLCζ and whether or not it is the only sperm factor in mammalian sperm.

Global loss of leucine carboxyl methyltransferase-1 causes severe defects in fetal liver hematopoiesis.

Leucine carboxyl methyltransferase-1 (LCMT-1) methylates the C-terminal leucine α-carboxyl group of the catalytic subunits of the protein phosphatase 2A (PP2A) subfamily of protein phosphatases, PP2Ac, PP4c, and PP6c. LCMT-1 differentially regulates the formation and function of a subset of the heterotrimeric complexes that PP2A and PP4 form with their regulatory subunits. Global LCMT-1 knockout causes embryonic lethality in mice, but LCMT-1 function in development is unknown. In this study, we analyzed the effects of global LCMT-1 loss on embryonic development. LCMT-1 knockout causes loss of PP2Ac methylation, indicating that LCMT-1 is the sole PP2Ac methyltransferase. PP2A heterotrimers containing the Bα and Bδ B-type subunits are dramatically reduced in whole embryos, and the steady-state levels of PP2Ac and the PP2A structural A subunit are also down ∼30%. Strikingly, global loss of LCMT-1 causes severe defects in fetal hematopoiesis and usually death by embryonic day 16.5. Fetal livers of homozygous lcmt-1 knockout embryos display hypocellularity, elevated apoptosis, and greatly reduced numbers of hematopoietic stem and progenitor cell-enriched Kit+Lin-Sca1+ cells. The percent cycling cells and mitotic indices of WT and lcmt-1 knockout fetal liver cells are similar, suggesting that hypocellularity may be due to a combination of apoptosis and/or defects in specification, self-renewal, or survival of stem cells. Indicative of a possible intrinsic defect in stem cells, noncompetitive and competitive transplantation experiments reveal that lcmt-1 loss causes a severe multilineage hematopoietic repopulating defect. Therefore, this study reveals a novel role for LCMT-1 as a key player in fetal liver hematopoiesis.

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iß pathway activation.

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras-/-) and control wild-type (H -ras+/+) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iß protein expression in H -ras-/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras-/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3ß-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iß overexpression in H -ras-/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iß overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iß pathway activation.

From masochistic enzymology to mechanistic physiology and disease.

The pioneering work of Eugene Kennedy in the 1950s established the choline pathway for phosphatidylcholine (PC) biosynthesis. However, the regulation of PC biosynthesis was poorly understood at that time. When I started my lab at the University of British Columbia in the 1970s, this was the focus of my research. This article provides my reflections on these studies that began with enzymology and the use of cultured mammalian cells, and progressed to utilize the techniques of molecular biology and gene-targeted mice. The research in my lab and others demonstrated that the regulated and rate-limiting step in the choline pathway for PC biosynthesis was catalyzed by CTP:phosphocholine cytidylyltransferase. This enzyme is regulated by its movement from a soluble form (largely in the nucleus) to a membrane-associated form where the enzyme becomes activated. Gene targeting in mice subsequently demonstrated that this gene is essential for development of mouse embryos. The other mammalian pathway for PC biosynthesis is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) that converts phosphatidylethanolamine to PC. Understanding of the regulation and function of the integral membrane protein PEMT was improved when the enzyme was purified (a masochistic endeavor) in 1987, leading to the cloning of the Pemt cDNA. Generation of knock-out mice that lacked PEMT showed that they were protected from atherosclerosis, diet-induced obesity, and insulin resistance. The protection from atherosclerosis appears to be due to decreased secretion of lipoproteins from the liver. We continue to investigate the mechanism(s) by which Pemt-/- mice are protected from weight gain and insulin resistance.

The deubiquitinating enzyme Usp14 allosterically inhibits multiple proteasomal activities and ubiquitin-independent proteolysis.

The proteasome-associated deubiquitinating enzyme Usp14/Ubp6 inhibits protein degradation by catalyzing substrate deubiquitination and by poorly understood allosteric actions. However, upon binding a ubiquitin chain, Usp14 enhances proteasomal degradation by stimulating ATP and peptide degradation. These studies were undertaken to clarify these seemingly opposite regulatory roles of Usp14 and their importance. To learn how the presence of Usp14 on 26S proteasomes influences its different activities, we compared enzymatic and regulatory properties of 26S proteasomes purified from wild-type mouse embryonic fibroblast cells and those lacking Usp14. The proteasomes lacking Usp14 had higher basal peptidase activity than WT 26S, and this activity was stimulated to a greater extent by adenosine 5'-O-(thiotriphosphate) (ATPγS) than with WT particles. These differences were clear even though Usp14 is present on only a minor fraction (30-40%) of the 26S in WT mouse embryonic fibroblast cells. Addition of purified Usp14 to the WT and Usp14-defficient proteasomes reduced both their basal peptidase activity and the stimulation by ATPγS. Usp14 inhibits these processes allosterically because a catalytically inactive Usp14 mutant also inhibited them. Proteasomes lacking Usp14 also exhibited greater deubiquitinating activity by Rpn11 and greater basal ATPase activity than WT particles. ATP hydrolysis by WT proteasomes is activated if they bind a ubiquitinated protein, which is loosely folded. Surprisingly, proteasomes lacking Usp14 could be activated by such proteins even without a ubiquitin chain present. Furthermore, proteasomes lacking Usp14 are much more active in degrading non-ubiquitinated proteins (e.g. Sic1) than WT particles. Thus, without a ubiquitinated substrate present, Usp14 suppresses multiple proteasomal activities, especially basal ATP consumption and degradation of non-ubiquitinated proteins. These allosteric effects thus reduce ATP hydrolysis by inactive proteasomes and nonspecific proteolysis and enhance proteasomal specificity for ubiquitinated proteins.

GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis.

Microvascular endothelial cells (ECs) are increasingly recognized as organ-specific gatekeepers of their microenvironment. Microvascular ECs instruct neighboring cells in their organ-specific vascular niches through angiocrine factors, which include secreted growth factors (angiokines), extracellular matrix molecules, and transmembrane proteins. However, the molecular regulators that drive organ-specific microvascular transcriptional programs and thereby regulate angiodiversity are largely elusive. In contrast to other ECs, which form a continuous cell layer, liver sinusoidal ECs (LSECs) constitute discontinuous, permeable microvessels. Here, we have shown that the transcription factor GATA4 controls murine LSEC specification and function. LSEC-restricted deletion of Gata4 caused transformation of discontinuous liver sinusoids into continuous capillaries. Capillarization was characterized by ectopic basement membrane deposition, formation of a continuous EC layer, and increased expression of VE-cadherin. Correspondingly, ectopic expression of GATA4 in cultured continuous ECs mediated the downregulation of continuous EC-associated transcripts and upregulation of LSEC-associated genes. The switch from discontinuous LSECs to continuous ECs during embryogenesis caused liver hypoplasia, fibrosis, and impaired colonization by hematopoietic progenitor cells, resulting in anemia and embryonic lethality. Thus, GATA4 acts as master regulator of hepatic microvascular specification and acquisition of organ-specific vascular competence, which are indispensable for liver development. The data also establish an essential role of the hepatic microvasculature in embryonic hematopoiesis.

INPP5E regulates phosphoinositide-dependent cilia transition zone function.

Human ciliopathies, including Joubert syndrome (JBTS), arise from cilia dysfunction. The inositol polyphosphate 5-phosphatase INPP5E localizes to cilia and is mutated in JBTS. Murine Inpp5e ablation is embryonically lethal and recapitulates JBTS, including neural tube defects and polydactyly; however, the underlying defects in cilia signaling and the function of INPP5E at cilia are still emerging. We report Inpp5e-/- embryos exhibit aberrant Hedgehog-dependent patterning with reduced Hedgehog signaling. Using mouse genetics, we show increasing Hedgehog signaling via Smoothened M2 expression rescues some Inpp5e-/- ciliopathy phenotypes and "normalizes" Hedgehog signaling. INPP5E's phosphoinositide substrates PI(4,5)P2 and PI(3,4,5)P3 accumulated at the transition zone (TZ) in Hedgehog-stimulated Inpp5e-/- cells, which was associated with reduced recruitment of TZ scaffolding proteins and reduced Smoothened levels at cilia. Expression of wild-type, but not 5-phosphatase-dead, INPP5E restored TZ molecular organization and Smoothened accumulation at cilia. Therefore, we identify INPP5E as an essential point of convergence between Hedgehog and phosphoinositide signaling at cilia that maintains TZ function and Hedgehog-dependent embryonic development.

TET-mediated DNA demethylation controls gastrulation by regulating Lefty-Nodal signalling.

Mammalian genomes undergo epigenetic modifications, including cytosine methylation by DNA methyltransferases (DNMTs). Oxidation of 5-methylcytosine by the Ten-eleven translocation (TET) family of dioxygenases can lead to demethylation. Although cytosine methylation has key roles in several processes such as genomic imprinting and X-chromosome inactivation, the functional significance of cytosine methylation and demethylation in mouse embryogenesis remains to be fully determined. Here we show that inactivation of all three Tet genes in mice leads to gastrulation phenotypes, including primitive streak patterning defects in association with impaired maturation of axial mesoderm and failed specification of paraxial mesoderm, mimicking phenotypes in embryos with gain-of-function Nodal signalling. Introduction of a single mutant allele of Nodal in the Tet mutant background partially restored patterning, suggesting that hyperactive Nodal signalling contributes to the gastrulation failure of Tet mutants. Increased Nodal signalling is probably due to diminished expression of the Lefty1 and Lefty2 genes, which encode inhibitors of Nodal signalling. Moreover, reduction in Lefty gene expression is linked to elevated DNA methylation, as both Lefty-Nodal signalling and normal morphogenesis are largely restored in Tet-deficient embryos when the Dnmt3a and Dnmt3b genes are disrupted. Additionally, a point mutation in Tet that specifically abolishes the dioxygenase activity causes similar morphological and molecular abnormalities as the null mutation. Taken together, our results show that TET-mediated oxidation of 5-methylcytosine modulates Lefty-Nodal signalling by promoting demethylation in opposition to methylation by DNMT3A and DNMT3B. These findings reveal a fundamental epigenetic mechanism featuring dynamic DNA methylation and demethylation crucial to regulation of key signalling pathways in early body plan formation.

Moderate folic acid supplementation and MTHFD1-synthetase deficiency in mice, a model for the R653Q variant, result in embryonic defects and abnormal placental development.

BACKGROUND: Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. OBJECTIVES: We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. DESIGN: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. RESULTS: The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet × maternal genotype, P = 0.0016; diet × embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S+/- embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. CONCLUSIONS: Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater incidence of defects in embryos. Although maternal circulating methylTHF was higher, it may not have reached the embryos because of abnormal placental development; abnormal placentas were observed predominantly in abnormally developed embryos. These findings have implications for women with high folate intakes, particularly if they are polymorphic for MTHFD1 R653Q.

Leucine Carboxyl Methyltransferase 1 (LCMT-1) Methylates Protein Phosphatase 4 (PP4) and Protein Phosphatase 6 (PP6) and Differentially Regulates the Stable Formation of Different PP4 Holoenzymes.

The protein phosphatase 2A (PP2A) subfamily of phosphatases, PP2A, PP4, and PP6, are multifunctional serine/threonine protein phosphatases involved in many cellular processes. Carboxyl methylation of the PP2A catalytic subunit (PP2Ac) C-terminal leucine is regulated by the opposing activities of leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) and regulates PP2A holoenzyme formation. The site of methylation on PP2Ac is conserved in the catalytic subunits of PP4 and PP6, and PP4 is also methylated on that site, but the identities of the methyltransferase enzyme for PP4 are not known. Whether PP6 is methylated is also not known. Here we use antibodies specific for the unmethylated phosphatases to show that PP6 is carboxyl-methylated and that LCMT-1 is the major methyltransferase for PP2A, PP4, and PP6 in mouse embryonic fibroblasts (MEFs). Analysis of PP2A and PP4 complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) indicates that PP4 holoenzyme complexes, like those of PP2A, are differentially regulated by LCMT-1, with the PP4 regulatory subunit 1 (PP4R1)-containing PP4 complex being the most dramatically affected by the LCMT-1 loss. MEFs derived from LCMT-1 knock-out mouse embryos have reduced levels of PP2A B regulatory subunit and PP4R1 relative to control MEFs, indicating that LCMT-1 is important for maintaining normal levels of these subunits. Finally, LCMT-1 homozygous knock-out MEFs exhibited hyperphosphorylation of HDAC3, a reported target of the methylation-dependent PP4R1-PP4c complex. Collectively, our data suggest that LCMT-1 coordinately regulates the carboxyl methylation of PP2A-related phosphatases and, consequently, their holoenzyme assembly and function.

Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers.

Drug resistance hinder most cancer chemotherapies and leads to disease recurrence and poor survival of patients. Resistance of cancer cells towards apoptosis is the major cause of these symptomatic behaviours. Here, we showed that isoquinoline alkaloids, including liensinine, isoliensinine, dauricine, cepharanthine and hernandezine, putatively induce cytotoxicity against a repertoire of cancer cell lines (HeLa, A549, MCF-7, PC3, HepG2, Hep3B and H1299). Proven by the use of apoptosis-resistant cellular models and autophagic assays, such isoquinoline alkaloid-induced cytotoxic effect involves energy- and autophagy-related gene 7 (Atg7)-dependent autophagy that resulted from direct activation of AMP activated protein kinase (AMPK). Hernandezine possess the highest efficacy in provoking such cell death when compared with other examined compounds. We confirmed that isoquinoline alkaloid is structurally varied from the existing direct AMPK activators. In conclusion, isoquinoline alkaloid is a new class of compound that induce autophagic cell death in drug-resistant fibroblasts or cancers by exhibiting its direct activation on AMPK.

Salicylate activates AMPK and synergizes with metformin to reduce the survival of prostate and lung cancer cells ex vivo through inhibition of de novo lipogenesis.

Aspirin, the pro-drug of salicylate, is associated with reduced incidence of death from cancers of the colon, lung and prostate and is commonly prescribed in combination with metformin in individuals with type 2 diabetes. Salicylate activates the AMP-activated protein kinase (AMPK) by binding at the A-769662 drug binding site on the AMPK ß1-subunit, a mechanism that is distinct from metformin which disrupts the adenylate charge of the cell. A hallmark of many cancers is high rates of fatty acid synthesis and AMPK inhibits this pathway through phosphorylation of acetyl-CoA carboxylase (ACC). It is currently unknown whether targeting the AMPK-ACC-lipogenic pathway using salicylate and/or metformin may be effective for inhibiting cancer cell survival. Salicylate suppresses clonogenic survival of prostate and lung cancer cells at therapeutic concentrations achievable following the ingestion of aspirin (<1.0 mM); effects not observed in prostate (PNT1A) and lung (MRC-5) epithelial cell lines. Salicylate concentrations of 1 mM increased the phosphorylation of ACC and suppressed de novo lipogenesis and these effects were enhanced with the addition of clinical concentrations of metformin (100 µM) and eliminated in mouse embryonic fibroblasts (MEFs) deficient in AMPK ß1. Supplementation of media with fatty acids and/or cholesterol reverses the suppressive effects of salicylate and metformin on cell survival indicating the inhibition of de novo lipogenesis is probably important. Pre-clinical studies evaluating the use of salicylate based drugs alone and in combination with metformin to inhibit de novo lipogenesis and the survival of prostate and lung cancers are warranted.

Baicalin promotes embryo adhesion and implantation by upregulating fucosyltransferase IV (FUT4) via Wnt/beta-catenin signaling pathway.

Glycosylation plays a significant role in determining the receptivity of the uterine endometrium to embryo. Fucosyltransferase IV (FUT4) is expressed stage-specifically in the uterine endometrium of mammalians, and considered as a marker of the endometrial receptivity. Baicalin, a monomer of flavonoids, is known to have functions in improving reproduction. However, the mechanism by which baicalin regulates the expression of FUT4 in embryo-endometrium adhesion remains unclear. Our results showed that baicalin significantly increased FUT4 mRNA and protein expression levels both in human endometrial cells and mouse endometrial tissue, and consistently elevated embryo adhesion rate during implantation in vitro and embryonic implantation competence in pregnant mouse. This study suggests that baicalin facilitates endometrial reproduction via elevating FUT4 expression through Wnt/ß-catenin signaling pathway.