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1.
Hum Mol Genet ; 26(20): 4011-4027, 2017 10 15.
Article in English | MEDLINE | ID: mdl-29016860

ABSTRACT

PLEKHA7, a gene recently associated with primary angle closure glaucoma (PACG), encodes an apical junctional protein expressed in components of the blood aqueous barrier (BAB). We found that PLEKHA7 is down-regulated in lens epithelial cells and in iris tissue of PACG patients. PLEKHA7 expression also correlated with the C risk allele of the sentinel SNP rs11024102 with the risk allele carrier groups having significantly reduced PLEKHA7 levels compared to non-risk allele carriers. Silencing of PLEKHA7 in human immortalized non-pigmented ciliary epithelium (h-iNPCE) and primary trabecular meshwork cells, which are intimately linked to BAB and aqueous humor outflow respectively, affected actin cytoskeleton organization. PLEKHA7 specifically interacts with GTP-bound Rac1 and Cdc42, but not RhoA, and the activation status of the two small GTPases is linked to PLEKHA7 expression levels. PLEKHA7 stimulates Rac1 and Cdc42 GTP hydrolysis, without affecting nucleotide exchange, identifying PLEKHA7 as a novel Rac1/Cdc42 GAP. Consistent with the regulatory role of Rac1 and Cdc42 in maintaining the tight junction permeability, silencing of PLEKHA7 compromises the paracellular barrier between h-iNPCE cells. Thus, downregulation of PLEKHA7 in PACG may affect BAB integrity and aqueous humor outflow via its Rac1/Cdc42 GAP activity, thereby contributing to disease etiology.


Subject(s)
Carrier Proteins/genetics , Glaucoma, Angle-Closure/genetics , cdc42 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/genetics , Blood-Aqueous Barrier/metabolism , Carrier Proteins/metabolism , Cell Movement/genetics , Epithelial Cells/metabolism , Genetic Predisposition to Disease , Glaucoma, Angle-Closure/metabolism , Glaucoma, Angle-Closure/pathology , Humans , Intercellular Junctions/metabolism , Iris/metabolism , Iris/pathology , Polymorphism, Single Nucleotide , Tight Junctions/metabolism , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism
2.
Invest Ophthalmol Vis Sci ; 55(6): 3833-41, 2014 May 06.
Article in English | MEDLINE | ID: mdl-24801512

ABSTRACT

PURPOSE: The role of the recently identified primary angle closure glaucoma (PACG) susceptibility gene, pleckstrin homology domain containing, family A member 7 (PLEKHA7), in PACG is unknown. PLEKHA7 associates with apical junctional complexes (AJCs) and is thus implicated in paracellular fluid regulation. We aimed to determine PLEKHA7's localization in the eye and its association with AJCs to elucidate its potential role in PACG. METHODS: Total RNA from ocular tissues was isolated and analyzed by real-time PCR. Frozen and paraffin-embedded human globes were sectioned and used for immunohistochemistry and immunofluorescence analysis. RESULTS: Specific PLEKHA7 expression was found in the muscles, vascular endothelium, and epithelium of the iris, ciliary body and ciliary processes, trabecular meshwork (TM), and choroid. PLEKHA7 expression in musculature and vascular endothelium was confirmed with smooth muscle marker, SMA, and endothelium marker, PECAM-1, respectively. At the above sites, PLEKHA7 colocalization was seen with adherens junction markers (E-cadherin and ß-catenin) and tight junction markers (ZO-1). CONCLUSIONS: Specific localization of PLEKHA7 was found within PACG-related structures (iris, ciliary body, and choroid) and blood-aqueous barrier (BAB) structures (posterior iris epithelium, nonpigmented ciliary epithelium, iris and ciliary body microvasculature). The association of PLEKHA7 with AJCs in the eye suggests a potential role for PLEKHA7 in PACG via fluidic regulation. Novel expression of PLEKHA7 was also seen in the ocular smooth muscles and vascular endothelia.


Subject(s)
Carrier Proteins/genetics , Endothelial Cells/metabolism , Epithelial Cells/metabolism , Gene Expression Regulation/physiology , Genetic Predisposition to Disease , Glaucoma, Angle-Closure/genetics , Intercellular Junctions/metabolism , Actins/metabolism , Biomarkers/metabolism , Carrier Proteins/metabolism , Cells, Cultured , Ciliary Body/blood supply , Ciliary Body/pathology , Fluorescent Antibody Technique, Indirect , Glaucoma, Angle-Closure/metabolism , Glaucoma, Angle-Closure/pathology , Humans , Immunohistochemistry , Iris/blood supply , Iris/pathology , Microscopy, Confocal , Muscle, Smooth/metabolism , Plasmids , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Real-Time Polymerase Chain Reaction , Transfection , Zonula Occludens-1 Protein/metabolism
3.
Cell Cycle ; 8(5): 773-9, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19221488

ABSTRACT

Many biological activities naturally oscillate. Here, we show that the NAD(+)/NADH ratios (redox status) fluctuate during mammalian cell cycle, with the S-phase redox status being the least oxidative. The S-phase NAD(+)/NADH redox status gates histone expression and S-phase progression, and may provide a genome protection mechanism during S-phase DNA replication as implicated in yeast. Accordingly, perturbing the cellular redox inhibits histone expression and leads to S-phase arrest. We propose that the S-phase NAD(+)/NADH redox status constitutes a redox signaling, which along with the cyclin E/cdk2 signaling regulates histone expression and S-phase progression.


Subject(s)
Histones/metabolism , NAD/metabolism , S Phase , Cell Cycle , Cell Line, Tumor , Cyclin E/genetics , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , HeLa Cells , Histones/genetics , Humans , Oxidation-Reduction , Signal Transduction
4.
Mol Endocrinol ; 23(6): 932-42, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19246513

ABSTRACT

Eukaryotic cells sense extracellular glucose concentrations via diverse mechanisms to regulate the expression of genes involved in metabolic control. One such example is the tight correlation between the expression of thioredoxin-interacting protein (Txnip) and extracellular glucose levels. In this report, we show that the transcription of the Txnip gene is induced by adenosine-containing molecules, of which an intact adenosine moiety is necessary and sufficient. Txnip promoter contains a carbohydrate response element, which mediates the induction of Txnip expression by these molecules in a glucose-dependent manner. Max-like protein X and MondoA are transcription factors previously shown to stimulate glucose-dependent Txnip expression and are shown here to convey stimulatory signals from extracellular adenosine-containing molecules to the Txnip promoter. The regulatory role of these molecules may be exerted via amplifying glucose signaling. Hence, this revelation may pave the way for interventions aimed toward metabolic disorders resulting from abnormal glucose homeostasis.


Subject(s)
Adenosine Triphosphate/pharmacology , Adenosine/pharmacology , Carrier Proteins/genetics , Glucose/metabolism , NAD/pharmacology , Signal Transduction/drug effects , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Carrier Proteins/metabolism , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Gene Expression Regulation/drug effects , Humans , Protein Transport/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Response Elements/genetics , Thioredoxins/metabolism , Time Factors , Transcription, Genetic/drug effects
5.
J Biol Chem ; 283(40): 26894-901, 2008 Oct 03.
Article in English | MEDLINE | ID: mdl-18682386

ABSTRACT

S-phase transcription of the histone 2B (H2B) gene is dependent on Octamer-binding factor 1 (Oct-1) and Oct-1 Co-Activator in S-phase (OCA-S), a protein complex comprising glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase (p38/GAPDH and p36/LDH) along with other components. H2B transcription in vitro is modulated by NAD(H). This potentially links the cellular redox status to histone expression. Here, we show that H2B transcription requires a proper NAD(+)/NADH redox status in vitro and in vivo. Therefore, perturbing a properly balanced redox impairs H2B transcription. A redox-modulated direct p38/GAPDH-Oct-1 interaction nucleates the occupancy of the H2B promoter by the OCA-S complex, in which p36/LDH plays a critical role in the hierarchical organization of the complex. As for p38/GAPDH, p36/LDH is essential for the OCA-S function in vivo, and OCA-S-associated p36/LDH possesses an LDH enzyme activity that impacts H2B transcription. These studies suggest that the cellular redox status (metabolic states) can directly feedback to gene switching in higher eukaryotes as is commonly observed in prokaryotes.


Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation/physiology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Histones/biosynthesis , L-Lactate Dehydrogenase/metabolism , Nitrosamines/metabolism , Octamer Transcription Factor-1/metabolism , Transcription Factors/metabolism , Animals , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , HeLa Cells , Histones/genetics , Humans , L-Lactate Dehydrogenase/genetics , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Octamer Transcription Factor-1/genetics , Oxidation-Reduction , Promoter Regions, Genetic/physiology , S Phase/physiology , Transcription Factors/genetics , Transcription, Genetic/physiology , Xenopus
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