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Nat Commun ; 12(1): 5550, 2021 09 21.
Article En | MEDLINE | ID: mdl-34548496

Understanding human development is of fundamental biological and clinical importance. Despite its significance, mechanisms behind human embryogenesis remain largely unknown. Here, we attempt to model human early embryo development with expanded pluripotent stem cells (EPSCs) in 3-dimensions. We define a protocol that allows us to generate self-organizing cystic structures from human EPSCs that display some hallmarks of human early embryogenesis. These structures mimic polarization and cavitation characteristic of pre-implantation development leading to blastocyst morphology formation and the transition to post-implantation-like organization upon extended culture. Single-cell RNA sequencing of these structures reveals subsets of cells bearing some resemblance to epiblast, hypoblast and trophectoderm lineages. Nevertheless, significant divergences from natural blastocysts persist in some key markers, and signalling pathways point towards ways in which morphology and transcriptional-level cell identities may diverge in stem cell models of the embryo. Thus, this stem cell platform provides insights into the design of stem cell models of embryogenesis.

Blastocyst/cytology , Cell Culture Techniques , Embryo, Mammalian/cytology , Embryonic Development/genetics , Models, Biological , Pluripotent Stem Cells/cytology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Biomarkers/metabolism , Blastocyst/metabolism , Cell Lineage/genetics , Embryo, Mammalian/anatomy & histology , Embryo, Mammalian/metabolism , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/metabolism , Gene Expression , Humans , Phospholipase C beta/genetics , Phospholipase C beta/metabolism , Pluripotent Stem Cells/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , SOXF Transcription Factors/genetics , SOXF Transcription Factors/metabolism , Sequence Analysis, RNA , Single-Cell Analysis
Int J Mol Sci ; 22(11)2021 May 30.
Article En | MEDLINE | ID: mdl-34070808

Metabotropic glutamate receptor subtype 5 (mGlu5) is implicated in the pathophysiology of Alzheimer´s disease (AD). However, its alteration at the subcellular level in neurons is still unexplored. Here, we provide a quantitative description on the expression and localisation patterns of mGlu5 in the APP/PS1 model of AD at 12 months of age, combining immunoblots, histoblots and high-resolution immunoelectron microscopic approaches. Immunoblots revealed that the total amount of mGlu5 protein in the hippocampus, in addition to downstream molecules, i.e., Gq/11 and PLCß1, was similar in both APP/PS1 mice and age-matched wild type mice. Histoblots revealed that mGlu5 expression in the brain and its laminar expression in the hippocampus was also unaltered. However, the ultrastructural techniques of SDS-FRL and pre-embedding immunogold demonstrated that the subcellular localisation of mGlu5 was significantly reduced along the neuronal surface of hippocampal principal cells, including CA1 pyramidal cells and DG granule cells, in APP/PS1 mice at 12 months of age. The decrease in the surface localisation of mGlu5 was accompanied by an increase in its frequency at intracellular sites in the two neuronal populations. Together, these data demonstrate, for the first time, a loss of mGlu5 at the plasma membrane and accumulation at intracellular sites in different principal cells of the hippocampus in APP/PS1 mice, suggesting an alteration of the excitability and synaptic transmission that could contribute to the cognitive dysfunctions in this AD animal model. Further studies are required to elucidate the specificity of mGlu5-associated molecules and downstream signalling pathways in the progression of the pathology.

Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Cell Membrane/metabolism , Hippocampus/metabolism , Pyramidal Cells/metabolism , Receptor, Metabotropic Glutamate 5/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/metabolism , Animals , Cell Membrane/pathology , Disease Models, Animal , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Gene Expression Regulation , Hippocampus/pathology , Humans , Male , Mice , Mice, Transgenic , Phospholipase C beta/genetics , Phospholipase C beta/metabolism , Presenilin-1/genetics , Presenilin-1/metabolism , Pyramidal Cells/pathology , Receptor, Metabotropic Glutamate 5/metabolism , Synaptic Transmission
Cells ; 10(5)2021 05 04.
Article En | MEDLINE | ID: mdl-34064422

Inhibition of the RAF-MEK1/2-ERK signaling pathway is an ideal strategy for treating cancers with NRAS or BRAF mutations. However, the development of resistance due to incomplete inhibition of the pathway and activation of compensatory cell proliferation pathways is a major impediment of the targeted therapy. The anthrax lethal toxin (LT), which cleaves and inactivates MEKs, is a modifiable biomolecule that can be delivered selectively to tumor cells and potently kills various tumor cells. However, resistance to LT and the mechanism involved are yet to be explored. Here, we show that LT, through inhibiting MEK1/2-ERK activation, inhibits the proliferation of cancer cells with NRAS/BRAF mutations. Among them, the human colorectal tumor HT-29 and murine melanoma B16-BL6 cells developed resistance to LT in 2 to 3 days of treatment. These resistant cells activated AKT through a histone deacetylase (HDAC) 8-dependent pathway. Using an Affymetrix microarray, followed by qPCR validation, we identified that the differential expression of the phospholipase C-ß1 (PLCB1) and squamous cell carcinoma-1 (DESC1) played an important role in HDAC8-mediated AKT activation and resistance to MEK1/2-ERK inhibition. By using inhibitors, small interference RNAs and/or expression vectors, we found that the inhibition of HDAC8 suppressed PLCB1 expression and induced DESC1 expression in the resistant cells, which led to the inhibition of AKT and re-sensitization to LT and MEK1/2 inhibition. These results suggest that targeting PLCB1 and DESC1 is a novel strategy for inhibiting the resistance to MEK1/2 inhibition.

Drug Resistance, Neoplasm , Histone Deacetylases/metabolism , Membrane Proteins/metabolism , Phospholipase C beta/metabolism , Repressor Proteins/metabolism , Serine Endopeptidases/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antigens, Bacterial/toxicity , Bacterial Toxins/toxicity , Cell Line, Tumor , Cell Proliferation/drug effects , HT29 Cells , Humans , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/metabolism , Membrane Proteins/genetics , Mice , Phospholipase C beta/genetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Serine Endopeptidases/genetics
Food Chem Toxicol ; 149: 111970, 2021 Mar.
Article En | MEDLINE | ID: mdl-33421459

Studies have shown that the central renin-angiotensin system is involved in neurological disorders. Our previous studies have demonstrated that angiotensin II receptor type 1 (AT1R) in the brain could be a potential target against methamphetamine (METH) use disorder. The present study was designed to investigate the underlying mechanisms of the inhibitory effect of AT1R on various behavioural effects of METH. We first examined the effect of AT1R antagonist, candesartan cilexetil (CAN), on behavioural and neurotoxic effects of METH. Furthermore, we studied the role of phospholipase C beta 1 (PLCß1) blockade behavioural and neurotoxic effects of METH. The results showed that CAN significantly attenuated METH-induced behavioral disorders and neurotoxicity associated with increased oxidative stress. AT1R and PLCß1 were significantly upregulated in vivo and in vitro. Inhibition of PLCß1 effectively alleviated METH-induced neurotoxicity and METH self-administration (SA) by central blockade of the PLCß1 involved signalling pathway. PLCß1 blockade significantly decreased the reinforcing and motivation effects of METH. PLCß1 involved signalling pathway, as well as a more specific role of PLCß1, involved the inhibitory effects of CAN on METH-induced behavioural dysfunction and neurotoxicity. Collectively, our findings reveal a novel role of PLCß1 in METH-induced neurotoxicity and METH use disorder.

Methamphetamine/administration & dosage , Methamphetamine/toxicity , Phospholipase C beta/metabolism , Signal Transduction/drug effects , Animals , Behavior, Animal/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Central Nervous System Stimulants , Gene Expression Regulation/drug effects , Humans , Male , Motor Activity , Neurotoxicity Syndromes , Phospholipase C beta/genetics , RNA Interference , RNA, Small Interfering , Rats , Rats, Sprague-Dawley
J Appl Genet ; 62(1): 107-113, 2021 Feb.
Article En | MEDLINE | ID: mdl-33131036

Auriculocondylar syndrome (ACS) is an ultra-rare disorder that arises from developmental defects of the first and second pharyngeal arches. Three subtypes of ACS have been described so far, i.e., ACS1 (MIM: 602483), ACS2 (MIM: 600810), and ACS3 (MIM: 131240). The majority of patients, however, are affected by ACS2, which results from the mutations in the PLCB4 gene. Herein, we have described an 8-year-old male patient presenting with ACS2 and summarized the molecular and phenotypic spectrum of the syndrome. We have also compared the clinical features of our case to three other previously described cases (one sporadic and two familial) harboring the same heterozygous missense variant c.1862G>A, p.Arg621His in the PLCB4 gene. The mutation was detected using whole-exome sequencing (WES). Due to low coverage of WES and suspicion of somatic mosaicism, the variant was additionally reassessed by deep targeted next-generation sequencing panel of genes related to the craniofacial disorders, and next confirmed by Sanger sequencing. ACS2 presents high intra- and interfamilial phenotypic heterogeneity that impedes reaching an exact clinical and molecular diagnosis. Thus, describing additional cases, carrying even the known mutation, but resulting in variable phenotypes, is essential for better understanding of such orphan Mendelian diseases.

Ear Diseases/genetics , Ear/abnormalities , Child , Humans , Male , Mutation , Pedigree , Phenotype , Phospholipase C beta/genetics
Mol Cell ; 80(6): 933-934, 2020 12 17.
Article En | MEDLINE | ID: mdl-33338407

Pfeil et al., (2020) examine the mechanism of Gi-stimulated Ca2+ release in cells and find an unexpected role for Gαq in Gßγ-dependent activation of phospholipase Cß (PLCß).

Calcium , GTP-Binding Protein alpha Subunits, Gq-G11 , Calcium/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Phospholipase C beta/genetics , Phospholipase C beta/metabolism , Signal Transduction
Nat Genet ; 52(12): 1314-1332, 2020 12.
Article En | MEDLINE | ID: mdl-33230300

Genetic studies of blood pressure (BP) to date have mainly analyzed common variants (minor allele frequency > 0.05). In a meta-analysis of up to ~1.3 million participants, we discovered 106 new BP-associated genomic regions and 87 rare (minor allele frequency ≤ 0.01) variant BP associations (P < 5 × 10-8), of which 32 were in new BP-associated loci and 55 were independent BP-associated single-nucleotide variants within known BP-associated regions. Average effects of rare variants (44% coding) were ~8 times larger than common variant effects and indicate potential candidate causal genes at new and known loci (for example, GATA5 and PLCB3). BP-associated variants (including rare and common) were enriched in regions of active chromatin in fetal tissues, potentially linking fetal development with BP regulation in later life. Multivariable Mendelian randomization suggested possible inverse effects of elevated systolic and diastolic BP on large artery stroke. Our study demonstrates the utility of rare-variant analyses for identifying candidate genes and the results highlight potential therapeutic targets.

Blood Pressure/genetics , Gene Frequency/genetics , Genetic Predisposition to Disease/genetics , Hypertension/genetics , GATA5 Transcription Factor/genetics , Genome-Wide Association Study , Genotype , Humans , Mutation/genetics , Phospholipase C beta/genetics , Polymorphism, Single Nucleotide/genetics
Mol Cell ; 80(6): 940-954.e6, 2020 12 17.
Article En | MEDLINE | ID: mdl-33202251

Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cß (PLCß) enzymes by G protein ßγ subunits from activated Gαi-Gßγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCß enzymes in living cells. We find that the Gαi-Gßγ-PLCß-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gßγ can bind to PLCß but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCß by Gßγ. This dependence of Gi-Gßγ-Ca2+ on Gαq places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCß enzymes.

GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein beta Subunits/genetics , GTP-Binding Protein gamma Subunits/genetics , Heterotrimeric GTP-Binding Proteins/genetics , Phospholipase C beta/genetics , Calcium/metabolism , Calcium Signaling/genetics , Cytosol/metabolism , HEK293 Cells , Humans , Protein Binding/genetics , Receptors, G-Protein-Coupled/genetics , Signal Transduction/genetics
BMC Genomics ; 21(1): 686, 2020 Oct 02.
Article En | MEDLINE | ID: mdl-33008286

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs playing vital roles in regulating posttranscriptional gene expression. Elucidating the expression regulation of miRNAs underlying pig testis development will contribute to a better understanding of boar fertility and spermatogenesis. RESULTS: In this study, miRNA expression profile was investigated in testes of Duroc and Meishan boars at 20, 75, and 270 days of age by high-throughput sequencing. Forty-five differentially expressed miRNAs were identified from testes of Duroc and Meishan boars before and after puberty. Integrated analysis of miRNA and mRNA profiles predicted many miRNA-mRNA pairs. Gene ontology and biological pathway analyses revealed that predicted target genes of ssc-mir-423-5p, ssc-mir-34c, ssc-mir-107, ssc-mir-196b-5p, ssc-mir-92a, ssc-mir-320, ssc-mir-10a-5p, and ssc-mir-181b were involved in sexual reproduction, male gamete generation, and spermatogenesis, and GnRH, Wnt, and MAPK signaling pathway. Four significantly differentially expressed miRNAs and their predicted target genes were validated by quantitative real-time polymerase chain reaction, and phospholipase C beta 1 (PLCß1) gene was verified to be a target of ssc-mir-423-5p. CONCLUSIONS: This study provides an insight into the functional roles of miRNAs in testis development and spermatogenesis and offers useful resources for understanding differences in sexual function development caused by the change in miRNAs expression between Duroc and Meishan boars.

Gene Regulatory Networks , MicroRNAs/genetics , RNA, Messenger/genetics , Swine/genetics , Testis/metabolism , Transcriptome , Animals , Cell Line , Male , MicroRNAs/metabolism , Phospholipase C beta/genetics , Phospholipase C beta/metabolism , RNA, Messenger/metabolism , Signal Transduction , Spermatogenesis , Swine/metabolism , Testis/cytology
FASEB J ; 34(11): 15400-15416, 2020 11.
Article En | MEDLINE | ID: mdl-32959428

MDS are characterized by anemia and transfusion requirements. Transfused patients frequently show iron overload that negatively affects hematopoiesis. Iron chelation therapy can be effective in these MDS cases, but the molecular consequences of this treatment need to be further investigated. That is why we studied the molecular features of iron effect and Deferasirox therapy on PI-PLCbeta1 inositide signaling, using hematopoietic cells and MDS samples. At baseline, MDS patients showing a positive response after iron chelation therapy displayed higher levels of PI-PLCbeta1/Cyclin D3/PKCalpha expression. During treatment, these responder patients, as well as hematopoietic cells treated with FeCl3 and Deferasirox, showed a specific reduction of PI-PLCbeta1/Cyclin D3/PKCalpha expression, indicating that this signaling pathway is targeted by Deferasirox. The treatment was also able to specifically decrease the production of ROS. This effect correlated with a reduction of IL-1A and IL-2, as well as Akt/mTOR phosphorylation. In contrast, cells exposed only to FeCl3 and cells from MDS patients refractory to Deferasirox showed a specific increase of ROS and PI-PLCbeta1/Cyclin D3/PKCalpha expression. All in all, our data show that PI-PLCbeta1 signaling is a target for iron-induced oxidative stress and suggest that baseline PI-PLCbeta1 quantification could predict iron chelation therapy response in MDS.

Cyclin D3/metabolism , Iron Overload/complications , Iron/adverse effects , Myelodysplastic Syndromes/therapy , Oxidative Stress/drug effects , Phospholipase C beta/metabolism , Protein Kinase C-alpha/metabolism , Aged , Blood Transfusion/statistics & numerical data , Cyclin D3/genetics , Deferasirox/pharmacology , Female , Gene Expression Regulation , Humans , Iron Chelating Agents/pharmacology , Male , Middle Aged , Myelodysplastic Syndromes/pathology , Phospholipase C beta/genetics , Phosphorylation , Protein Kinase C-alpha/genetics , Signal Transduction
Exp Cell Res ; 395(2): 112219, 2020 10 15.
Article En | MEDLINE | ID: mdl-32763246

Increasing evidence has shown that abnormal expression of XPO5 is found in many human cancers and acts as an oncoprotein in certain cancers. However, its functional role in hepatocellular carcinoma (HCC) remains unexplored. In our study, we found that XPO5 was highly expressed in HCC, which was associated with SUMO modification. Moreover, we found that XPO5 was SUMOylated by SUMO2 at K125. Functional experiments revealed that XPO5 SUMOylation could promote MHCC97H cell proliferation, migration and invasion. In addition, we found that the nuclear export of pre-miR-3184 was suppressed by SUMOylated XPO5. Moreover, PLCB1 was identified as the common target of miR-3184-5p and miR-3184-3p. The suppressed phenotype induced by miR-3184-5p and miR-3184-3p could be rescued by overexpression of PLCB1. Bioinformatics analysis showed that PLCB1 expression had a negative relationship with HCC patient survival. The inhibitory effects of MHCC97H cells resulted from abnormal XPO5 SUMO modification could be blocked by miR-3184 inhibitor or PLCB1 overexpression. In conclusion, our findings demonstrate a novel mechanism of XPO5 in HCC, that is, the SUMOylated XPO5 acts as an "oncogenic" role in MHCC97H cells proliferation, migration and invasion by controlling the nuclear-cytoplasm transportation of miR-3184, thus up-regulating PLCB1 expression.

Karyopherins/genetics , Liver Neoplasms/genetics , Carcinogenesis/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cell Proliferation/physiology , Gene Expression Regulation, Neoplastic/genetics , Humans , Liver Neoplasms/pathology , MicroRNAs/genetics , Phospholipase C beta/genetics , Sumoylation/genetics , Sumoylation/physiology
Genes (Basel) ; 11(5)2020 05 14.
Article En | MEDLINE | ID: mdl-32423003

Hanwoo, is the most popular native beef cattle in South Korea. Due to its extensive popularity, research is ongoing to enhance its carcass quality and marbling traits. In this study we conducted a haplotype-based genome-wide association study (GWAS) by constructing haplotype blocks by three methods: number of single nucleotide polymorphisms (SNPs) in a haplotype block (nsnp), length of genomic region in kb (Len) and linkage disequilibrium (LD). Significant haplotype blocks and genes associated with them were identified for carcass traits such as BFT (back fat thickness), EMA (eye Muscle area), CWT (carcass weight) and MS (marbling score). Gene-set enrichment analysis and functional annotation of genes in the significantly-associated loci revealed candidate genes, including PLCB1 and PLCB4 present on BTA13, coding for phospholipases, which might be important candidates for increasing fat deposition due to their role in lipid metabolism and adipogenesis. CEL (carboxyl ester lipase), a bile-salt activated lipase, responsible for lipid catabolic process was also identified within the significantly-associated haplotype block on BTA11. The results were validated in a different Hanwoo population. The genes and pathways identified in this study may serve as good candidates for improving carcass traits in Hanwoo cattle.

Cattle/genetics , Genome-Wide Association Study , Haplotypes/genetics , Adiposity , Animals , Body Weight , Cadaver , Gene Ontology , Lipase/genetics , Meat , Oculomotor Muscles/anatomy & histology , Phospholipase C beta/genetics , Polymorphism, Single Nucleotide , Software
Development ; 147(9)2020 05 13.
Article En | MEDLINE | ID: mdl-32317273

The ability of a muscle to break down and reform fibers is vital for development; however, if unregulated, abnormal muscle remodeling can occur, such as in the heart following cardiac infarction. To study how normal developmental remodeling is mediated, we used fluorescently tagged actin, mutant analyses, Ca2+ imaging and controlled Ca2+ release to determine the mechanisms regulating a conspicuous muscle change that occurs in Caenorhabditis elegans males. In hermaphrodites and larval males, the single cell anal depressor muscle, used for waste expulsion, contains bilateral dorsal-ventral sarcomeres. However, prior to male adulthood, the muscle sex-specifically remodels its sarcomeres anteriorly-posteriorly to promote copulation behavior. Although WNT signaling and calcineurin have been implicated separately in muscle remodeling, we unexpectedly found that they participate in the same pathway. We show that WNT signaling through Gαo and PLC-ß results in sustained Ca2+ release via IP3 and ryanodine receptors to activate calcineurin. These results highlight the utility of this new model in identifying additional molecules involved in muscle remodeling.

Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Calcineurin/metabolism , Phospholipase C beta/metabolism , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Calcineurin/genetics , Calcium/metabolism , Male , Phospholipase C beta/genetics , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/genetics , Signal Transduction/physiology
Eur J Med Genet ; 63(6): 103917, 2020 Jun.
Article En | MEDLINE | ID: mdl-32201334

Auriculocondylar syndrome (ARCND, MIM #614669, #602483, and #615706); also known as ''question-mark ear syndrome'' or ''dysgnathia complex'', is a rare craniofacial malformation of first and second branchial arches with a prevalence of <1/1,000,000. It is characterized by a distinctive auricular malformation (question mark ear (QME)) and highly variable mandibular anomalies. Variants found in PLCB4, GNAI3, and in EDN1 genes are responsible for >90% of tested ARCND patients. Whole exome sequencing in a multigenerational Egyptian kindred with high intrafamilial variability revealed a known heterozygous missense variant in PLCB4 (NM_000933.3:c.1862G>A:p.(Arg621His)). This report increases the number of molecularly characterized ARCND patients to 29 and emphasizes the highly variable clinical presentation within families.

Ear Diseases/genetics , Ear/abnormalities , Mutation , Phenotype , Phospholipase C beta/genetics , Adolescent , Adult , Child , Ear/pathology , Ear Diseases/pathology , Endothelin-1/genetics , Female , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Humans , Male , Pedigree
Int J Mol Sci ; 21(3)2020 Feb 10.
Article En | MEDLINE | ID: mdl-32050555

Cholesterol is a critical component of eukaryotic membranes, where it contributes to regulating transmembrane signaling, cell-cell interaction, and ion transport. Dysregulation of cholesterol levels in the brain may induce neurodegenerative diseases, such as Alzheimer's disease, Parkinson disease, and Huntington disease. We previously reported that augmenting membrane cholesterol level regulates ion channels by decreasing the level of phosphatidylinositol 4,5-bisphosphate (PIP2), which is closely related to ß-amyloid (Aß) production. In addition, cholesterol enrichment decreased PIP2 levels by increasing the expression of the ß1 isoform of phospholipase C (PLC) in cultured cells. In this study, we examined the effect of a high-cholesterol diet on phospholipase C (PLCß1) expression and PIP2 levels in rat brain. PIP2 levels were decreased in the cerebral cortex in rats on a high-cholesterol diet. Levels of PLCß1 expression correlated with PIP2 levels. However, cholesterol and PIP2 levels were not correlated, suggesting that PIP2 level is regulated by cholesterol via PLCß1 expression in the brain. Thus, there exists cross talk between cholesterol and PIP2 that could contribute to the pathogenesis of neurodegenerative diseases.

Brain/metabolism , Cholesterol/pharmacology , Diet, High-Fat/adverse effects , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phospholipase C beta/genetics , Animals , Cholesterol/metabolism , Male , Phospholipase C beta/metabolism , Rats , Rats, Sprague-Dawley
J Cell Mol Med ; 24(5): 3192-3202, 2020 03.
Article En | MEDLINE | ID: mdl-31975557

As a common complication of pregnancy, gestational hypoxia has been shown to predispose offspring to vascular dysfunction. Propionate, one of short-chain fatty acids, exerts cardioprotective effects via reducing blood pressure. This study examined whether prenatal hypoxia impaired propionate-stimulated large-conductance Ca2+ -activated K+ (BK) channel activities in vascular smooth muscle cells (VSMCs) of offspring. Pregnant rats were exposed to hypoxia (10.5% oxygen) and normoxia (21% oxygen) from gestational day 7-21. At 6 weeks of age, VSMCs in mesenteric arteries of offspring were analysed for BK channel functions and gene expressions. It was shown firstly that propionate could open significantly BK single channel in VSMCs in a concentration-dependent manner. Antagonists of G protein ßγ subunits and inositol trisphosphate receptor could completely suppress the activation of BK by propionate, respectively. Gαi/o and ryanodine receptor were found to participate in the stimulation on BK. Compared to the control, vasodilation and increments of BK NPo (the open probability) evoked by propionate were weakened in the offspring by prenatal hypoxia with down-regulated Gßγ and PLCß. It was indicated that prenatal hypoxia inhibited propionate-stimulated BK activities in mesenteric VSMCs of offspring via reducing expressions of Gßγ and PLCß, in which endoplasmic reticulum calcium release might be involved.

Hypoxia/drug therapy , Large-Conductance Calcium-Activated Potassium Channels/genetics , Pregnancy Complications, Cardiovascular/drug therapy , Prenatal Exposure Delayed Effects/drug therapy , Propionates/pharmacology , Animals , Blood Pressure/drug effects , Calcium Signaling/drug effects , Female , GTP-Binding Protein beta Subunits/genetics , Humans , Hypoxia/complications , Hypoxia/genetics , Hypoxia/pathology , Inositol 1,4,5-Trisphosphate Receptors/genetics , Mesenteric Arteries/drug effects , Mesenteric Arteries/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Phospholipase C beta/genetics , Pregnancy , Pregnancy Complications, Cardiovascular/genetics , Pregnancy Complications, Cardiovascular/pathology , Prenatal Exposure Delayed Effects/genetics , Prenatal Exposure Delayed Effects/metabolism , Prenatal Exposure Delayed Effects/pathology , Rats
Mol Med Rep ; 21(1): 420-428, 2020 01.
Article En | MEDLINE | ID: mdl-31746389

Malignant melanoma has the highest malignancy rate among all skin cancer and is characterized by an insidious onset, high invasion and poor patient prognosis. Yet, the mechanisms involved remain unclear and warrant further investigation. Based on bioinformatic analysis, phospholipase C ß2 (PLCB2) has been found to be correlated with melanoma growth. The present study was the first to demonstrate that PLCB2 is a key factor affecting melanoma proliferation and apoptosis. Here, microarray datasets from the publicly available Gene Expression Omnibus (GEO) database were employed, and gene set enrichment analysis (GSEA) was introduced to identify candidate transcription factors. PLCB2 was identified as a crucial gene in the protein­protein interaction (PPI) network. The expression of PLCB2 mRNA in various cancer lines was analyzed by reverse transcription­polymerase chain reaction (RT­PCR). In addition, the proliferation ability and apoptosis rate in human melanoma cells overexpressing or not overexpressing PLCB2 were assessed using colony formation assay, flow cytometry and the Cell Counting Kit­8 (CCK­8) assay. Cell viability and apoptosis­related factors, such as p53, Bcl­2, Bax and caspase­3 were significantly regulated. Knockdown of PLCB2 suppressed the activation of the Ras/Raf/MAPK signaling pathway. In conclusion, knockdown of PLCB2 suppressed cell viability and promoted cell apoptosis by activating the Ras/Raf/MAPK pathway. Thus, PLCB2 may utilized as a potential therapeutic target in patients with melanoma.

Cell Proliferation/genetics , Melanoma/genetics , Neoplasm Proteins/genetics , Phospholipase C beta/genetics , Apoptosis/genetics , Cell Line, Tumor , Cell Survival/genetics , Gene Expression Regulation, Neoplastic/genetics , Gene Knockdown Techniques , Genes, ras/genetics , Humans , MAP Kinase Kinase 1/genetics , Melanoma/pathology , Microarray Analysis , Protein Interaction Maps , Proto-Oncogene Proteins c-raf/genetics , Signal Transduction/genetics
Ophthalmic Res ; 63(3): 358-368, 2020.
Article En | MEDLINE | ID: mdl-31614358

BACKGROUND: The purpose of this study is to determine the mutation frequencies of key driver genes in uveal melanoma (UM) in Chinese patients and to detect associations between metastasis and the mutation of these genes. METHOD: A total of 85 patients with UM were enrolled in this study, including 18 patients with metastasis and 67 without metastasis. Sanger sequencing covering the mutational hotspot regions of the G protein subunit alpha Q (GNAQ), GNA11, splicing factor 3B subunit 1 (SF3B1), X-linked eukaryotic translation initiation factor 1A (EIF1AX), phospholipase C beta 4 (PLCB4) and cysteinyl leukotriene receptor 2 (CYSLTR2) genes was used to analyse the mutations in Chinese patients. RESULTS: The frequencies of GNAQ and GNA11 mutations in UM were 45% (38/85) and 35% (30/85) respectively. The frequencies of SF3B1 and EIF1AX mutations were 37% (31/85) and 9% (8/85) respectively. Only 2 mutations were detected in exon 4 of GNAQ, and no mutations were detected in exon 4 of GNA11. A novel mutation, c.627G>T (Q209H) in GNA11 was found. The detected mutations affecting SF3B1 were c.1873C>T (R625C), c.1874G>A (R625H) and c.1874G>T (R625L). The association between the mutations in SF3B1 and low risk of metastasis was statistically significant (OR 0.17, 95% CI 0.035-0.819). The mutations affecting EIF1AX were -23G>A (5'-UTR), c.5C>G (P2R), c.23G>A (G8Q), c.25G>C (G9A) and c.38_39GC>CT (R13P). No mutations were found in the PLCB4 and CYSLTR2 genes. Unfortunately, information on BRCA1-associated protein 1 could not be obtained. CONCLUSIONS: These data indicate that mutations in the PLCB4 and CYSLTR2 genes are rare in Chinese UM patients. The mutations in GNAQ, GNA11 and EIF1AX were not associated with metastasis, whereas SF3B1 mutations were correlated with low risk of metastasis and demonstrated a protective effect in UM patients in China.

Eukaryotic Initiation Factor-1/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , Melanoma/genetics , Mutation , Phospholipase C beta/genetics , Phosphoproteins/genetics , RNA Splicing Factors/genetics , Receptors, Leukotriene/genetics , Uveal Neoplasms/genetics , Adolescent , Adult , Aged , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , DNA Mutational Analysis , DNA, Neoplasm/genetics , Eukaryotic Initiation Factor-1/metabolism , Eye Neoplasms/diagnosis , Eye Neoplasms/genetics , Eye Neoplasms/metabolism , Female , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Humans , Male , Melanoma/diagnosis , Melanoma/metabolism , Middle Aged , Phospholipase C beta/metabolism , Phosphoproteins/metabolism , RNA Splicing Factors/metabolism , Receptors, Leukotriene/metabolism , Retrospective Studies , Uveal Neoplasms/diagnosis , Uveal Neoplasms/metabolism , Young Adult
Cardiovasc Pathol ; 44: 107160, 2020.
Article En | MEDLINE | ID: mdl-31759320

Chromogranin B and inositol 1,4,5-trisphosphate-associated calcium signaling leading to increased natriuretic peptide production has been described in cardiac hypertrophy. Here, we performed left anterior descending coronary artery ligation in rats as a model for systolic heart failure and examined protein and gene expression clusters in the infarcted and noninfarcted myocardium and moreover under treatment with metoprolol. We found that atrial natriuretic peptide gene transcription was significantly more elevated in the infarcted compared with the noninfarcted myocardium. Chromogranin B, which facilitates calcium release from internal stores through the inositol 1,4,5-trisphosphate receptor, was upregulated in both areas. Interestingly, angiotensin II receptor type 1 gene transcription was significantly upregulated in the infarcted and unchanged in the noninfarcted myocardium. Nuclear factor ĸappa B as a calcium-dependent transcription factor showed increased activity in the infarction zone. The ß-adrenergic axis does not seem to be involved, as metoprolol treatment did not have a significant impact on any of these results. We conclude that region-specific upregulation of angiotensin II receptor type 1 is a major factor for increased atrial natriuretic peptide production in the infarcted anterior wall. This effect is most likely achieved through inositol 1,4,5-trisphosphate-mediated cytosolic calcium increase and subsequent nuclear factor ĸappa B activation, which is a known transcription factor for natriuretic peptides.

Atrial Natriuretic Factor/metabolism , Heart Failure/metabolism , Myocardial Infarction/metabolism , Myocardium/metabolism , Receptor, Angiotensin, Type 1/metabolism , Adrenergic beta-1 Receptor Antagonists/pharmacology , Animals , Atrial Natriuretic Factor/genetics , Calcium Signaling , Chromogranin B/genetics , Chromogranin B/metabolism , Disease Models, Animal , Heart Failure/drug therapy , Heart Failure/genetics , Heart Failure/pathology , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Male , Metoprolol/pharmacology , Myocardial Infarction/drug therapy , Myocardial Infarction/genetics , Myocardial Infarction/pathology , Myocardium/pathology , NF-kappa B/metabolism , Phospholipase C beta/genetics , Phospholipase C beta/metabolism , Rats, Wistar , Receptor, Angiotensin, Type 1/genetics
Clin Genet ; 97(3): 477-482, 2020 03.
Article En | MEDLINE | ID: mdl-31883110

Biallelic mutations in the PLCB1 gene, encoding for a phospholipase C beta isoform strongly expressed in the brain, have been reported to cause infantile epileptic encephalopathy in only four children to date. We report here three additional patients to delineate the phenotypic and genotypic characteristics of the disease. Our three patients were one sporadic case with an intragenic homozygous deletion and two cousins with the homozygous p.(Arg222*) nonsense variant in PLCB1. These patients had severe to profound intellectual disability, epileptic spasms at age 3-5 months concomitant with developmental arrest or regression, other seizure types and drug-resistant epilepsy. With this report, we expand the clinical, radiologic and electroencephalographic knowledge about the extremely rare PLCB1-related encephalopathy. Since the first report in 2010, the overall number of reported patients with our additional patients is currently limited to seven. All seven patients had epileptic encephalopathy, mainly infantile spasms and 6/7 had profound intellectual disability, with one only being able to walk. Truncal hypotonia was the most frequent neurological sign, sometimes associated with pyramidal and/or extrapyramidal hypertonia of limbs. Microcephaly was inconstant. In conclusion, the phenotypical spectrum of PLCB1-related encephalopathy is relatively narrow, comprises infantile spasms and severe to profound intellectual disability, and does not seem to define a recognizable clinical entity.

Phospholipase C beta/genetics , Seizures/genetics , Spasms, Infantile/genetics , Brain/diagnostic imaging , Brain/pathology , Child , Child, Preschool , Female , Genotype , Homozygote , Humans , Infant , Intellectual Disability/genetics , Intellectual Disability/pathology , Male , Phenotype , Seizures/pathology , Sequence Deletion/genetics , Spasms, Infantile/diagnostic imaging , Spasms, Infantile/pathology