Levels of tight junction protein CLDND1 are regulated by microRNA-124 in the cerebellum of stroke-prone spontaneously hypertensive rats.

The claudin family shows organ- and tissue-specific expression of individual members. Deficiency or aberrant expression of distinct claudins has been reported to be associated with severe pathophysiological consequences. Claudin domain-containing 1 (CLDND1), also known as claudin-25, shows homology to this family of proteins. Furthermore, serum CLDND1-derived peptide antibody levels are elevated in patients with cerebral infarction, as compared with healthy controls. We previously reported that, in the adult murine brain, CLDND1 is abundantly expressed in the cerebellum in common sites of intracerebral hemorrhage, and CLDND1 levels are transiently decreased after hemorrhagic insult. However, regulation of CLDND1 expression levels in cerebrovascular disease is poorly studied, and most regulatory microRNAs remain to be defined. We assessed its expression level, according to the presence of early signs of cerebrovascular disease, in the brain of stroke-prone spontaneously hypertensive rats (SHRSPs) and investigated the microRNA regulation of Cldnd1 mRNA. We investigated the post-transcriptional regulation of Cldnd1 by examining the subcellular distribution of its mRNA and evaluating its translational regulation by microRNA in human brain endothelial cells (HBECs) and in the brain of SHRSPs. Using bioinformatics, we identified a conserved microRNA-124 (miR-124)-binding site in the 3'-untranslated region of Cldnd1 and demonstrated that miR-124 regulates the translation of Cldnd1 mRNA reporters in a sequence-specific manner in luciferase assays. HBECs transfected with an miR-124 mimic showed decreased levels of CLDND1 mRNA in reverse transcription quantitative PCR. miR-124 levels were markedly lower in SHRSP than in Wister Kyoto rat brains, whereas Cldnd1 mRNA and protein levels were significantly higher. In SHRSP brains, Cldnd1 mRNA levels increased with a decrease in miR-124. Therefore, by interacting with Cldnd1 mRNA, miR-124 influences CLDNL1 levels in the brain, thus playing a role in the development of cerebrovascular disease in SHRSPs.

Downregulation of Histone H3.3 Induces p53-Dependent Cellular Senescence in Human Diploid Fibroblasts.

Cellular senescence is an irreversible growth arrest that acts as a barrier to cancer initiation and progression. Histone alteration is one of the major events during replicative senescence. However, little is known about the function of H3.3 in cellular senescence. Here we found that the downregulation of H3.3 induced growth suppression with senescence-like phenotypes such as senescence-associated heterochromatin foci (SAHF) and ß-galactosidase (SA-ß-gal) activity. Furthermore, H3.3 depletion induced senescence-like phenotypes with the p53/p21-depedent pathway. In addition, we identified miR-22-3p, tumor suppressive miRNA, as an upstream regulator of the H3F3B (H3 histone, family 3B) gene which is the histone variant H3.3 and replaces conventional H3 in active genes. Therefore, our results reveal for the first time the molecular mechanisms for cellular senescence which are regulated by H3.3 abundance. Taken together, our studies suggest that H3.3 exerts functional roles in regulating cellular senescence and is a promising target for cancer therapy.

High-throughput evaluation of aryl hydrocarbon receptor-binding sites selected via chromatin immunoprecipitation-based screening in Hepa-1c1c7 cells stimulated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt) and binds to DNA. It has been shown that the binding of AhR to DNA depends on the dioxin response element (DRE) and controls xenobiotic-response genes. AhR-binding DNA fragments from mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD were once enriched in a chromatin immunoprecipitation (ChIP) DNA library and screened through a high-throughput southwestern chemistry-based enzyme-linked immunosorbent assay (SW-ELISA). After screening 1700 fragments, the ChIP-SW-ELISA screening strategy allowed us to isolate 77 fragments tightly interacting with AhR in the presence of TCDD. Only 39 of the 77 fragments appeared to contain a typical DRE, indicating that in some cases the DRE was dispensable for AhR-binding, while 75 fragments were located within promoter-distal regions. Genomic mapping of the 77 fragments enabled us to estimate 121 potential AhR targets including known targets such as Cyp1A1 and Cyp1B1, but only a limited number exhibited an altered expression dependent on TCDD. This study revealed the fact that TCDD-activated AhR frequently binds to promoter-distal regions even without a DRE and is not always involved in transcriptional regulation, suggesting that within the genome DNA-binding of AhR could take place often in many regions without cis-regulatory elements and might not be a key determinant to establish its regulatory function.

Bone morphogenetic protein 4 promotes craniofacial neural crest induction from human pluripotent stem cells.

Neural crest (NC) cells are a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. Cranial NC cells migrate to the branchial arches and give rise to the majority of the craniofacial region, whereas trunk and tail NC cells contribute to the heart, enteric ganglia of the gut, melanocytes, sympathetic ganglia, and adrenal chromaffin cells. Positional information is indispensable for the regulation of cranial or trunk and tail NC cells. However, the mechanisms underlying the regulation of positional information during human NC induction have yet to be fully elucidated. In the present study, supplementation of bone morphogenetic protein (BMP) 4 in defined serum-free culture conditions including fibroblast growth factor-2 and Wnt3a from day 8 after NC specification induced the expression of cranial NC markers, AP2alpha, MSX1, and DLX1, during NC cell differentiation from human pluripotent stem cells. On the other hand, the proportion of cells expressing p75(NTR) or HNK1 decreased compared with that of cells cultured without BMP4, whereas gene expression analysis demonstrated that the expression levels of cranial NC-associated genes increased in BMP4-treated NC cells. These BMP4-treated NC cells were capable of differentiation into osteocytes and chondrocytes. The results of the present study indicate that BMP4 regulates cranial positioning during NC development.

Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control.

Given the difficulties inherent in maintaining human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established standard criteria during expansion for research or therapeutic purposes. hPSC colony morphology is typically considered an important criterion, but it is not evaluated quantitatively. Thus, we designed an unbiased method to evaluate hPSC colony morphology. This method involves a combination of automated non-labelled live-cell imaging and the implementation of morphological colony analysis algorithms with multiple parameters. To validate the utility of the quantitative evaluation method, a parent cell line exhibiting typical embryonic stem cell (ESC)-like morphology and an aberrant hPSC subclone demonstrating unusual colony morphology were used as models. According to statistical colony classification based on morphological parameters, colonies containing readily discernible areas of differentiation constituted a major classification cluster and were distinguishable from typical ESC-like colonies; similar results were obtained via classification based on global gene expression profiles. Thus, the morphological features of hPSC colonies are closely associated with cellular characteristics. Our quantitative evaluation method provides a biological definition of 'hPSC colony morphology', permits the non-invasive monitoring of hPSC conditions and is particularly useful for detecting variations in hPSC heterogeneity.

Synergistic effects of FGF-2 and Activin A on early neural differentiation of human pluripotent stem cells.

Neural differentiation is an important target of human embryonic stem cells, which provide a source for cell-based therapy, developmental biology, and pharmaceutical research. Previous studies revealed that inhibition of the bone morphogenetic protein is required for neural induction from human embryonic stem cells. On the contrary, the functions of fibroblast growth factors and Activin/Nodal signaling are controversial. Fibroblast growth factor-2 and Activin/Nodal pathways exert divergent influences on human embryonic stem cell concerning the maintenance of both pluripotency and cellular differentiation. We hypothesized that the combination of fibroblast growth factor-2 and Activin A at various concentrations synergistically exerts diverse effects on cell differentiation. To determine the effects of fibroblast growth factor-2 and Activin A on cellular differentiation into neural lineages, we examined the expression of neural differentiation markers in human embryonic stem cells treated with fibroblast growth factor-2 and/or Activin A at various concentrations in a growth factor-defined serum-free medium in short-term culture. In this study, we provide evidence that fibroblast growth factor-2 and Activin A synergistically regulated the initiation of human embryonic stem cell differentiation into neural cell lineages even though human embryonic stem cells autonomously differentiate into neural cell lineages.

Protein kinase C-induced early growth response protein-1 binding to SNAIL promoter in epithelial-mesenchymal transition of human embryonic stem cells.

Epithelial-mesenchymal transition (EMT) has been thought to occur during early embryogenesis, and also the differentiation process of human embryonic stem (hES) cells. Spontaneous differentiation is sometimes observed at the peripheral of the hES cell colonies in conventional culture conditions, indicating that EMT occurs in hES cell culture. However, the triggering mechanism of EMT is not yet fully understood. The balance between self-renewal and differentiation of human pluripotent stem (hPS) cells is controlled by various signal pathways, including the fibroblast growth factor (FGF)-2. However, FGF-2 has a complex role for self-renewal of hES cells. FGF-2 activates phosphatidylinositol-3 kinase/AKT, mitogen-activated protein kinase/extracellular signal-regulated kinase-1/2 kinase, and also protein kinase C (PKC). Here, we showed that a PKC rapidly induced an early growth response protein-1 (EGR-1) in hES cells, which was followed by upregulation of EMT-related genes. Before the induction of EMT-related genes, EGR-1 was translocated into the nucleus, and then bound directly to the promoter region of SNAIL, which is a master regulator of EMT. SNAIL expression was attenuated by knockdown of EGR-1, but upregulated by ectopic expression of EGR-1. EGR-1 as the downstream signal of PKC might play a key role in EMT initiation during early differentiation of hES cells. This study would lead to a more robust understanding of the mechanisms underlying the balance between self-renewal and initiation of differentiation in hPS cells.

Protein kinase C regulates human pluripotent stem cell self-renewal.

BACKGROUND: The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3ß (GSK-3ß), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3ß. Addition of activin A increased phosphorylation of GSK-3ß and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, ß, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3ß was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells. CONCLUSIONS/SIGNIFICANCE: Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3ß. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells.

Aryl hydrocarbon receptor-mediated induction of the cytosolic phospholipase A(2)alpha gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse hepatoma Hepa-1c1c7 cells.

Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt). This complex binds to DNA. It has been shown that the AhR bonds to a DNA sequence called the dioxin response element (DRE), which controls the expression of battery genes. It is reported that TCDD releases arachidonic acid from membrane phospholipids via activation of phospholipase A(2)s (PLA(2)s) in various cell types. Recently, we demonstrated that the TCDD-activated AhR binds to the second intron of the Pla2g4a gene, which encodes cytosolic phospholipase A(2)alpha (cPLA(2)alpha), in mouse hepatoma Hepa-1c1c7 cells. This result suggests that Pla2g4a appears to be a target gene of the AhR. In the present study, we investigated whether the transcriptional regulation of Pla2g4a is dependent on the AhR in Hepa-1c1c7 cells. Treatment of the cells with TCDD increased mRNA expression of Pla2g4a and enzymatic activity of PLA(2,) while this increased expression was not observed in AhR-defective c12 cells. After transient transfection of an Ahr gene-expressing plasmid into the c12 cells, expression of Pla2g4a was increased by TCDD. These results indicate that Pla2g4a may be a novel target gene of the AhR, and its transcriptional induction is mediated through binding of the AhR to the second intron of Pla2g4a, although this target site does not have a typical DRE sequence.

myo-Inositol catabolism in Bacillus subtilis.

The iolABCDEFGHIJ operon of Bacillus subtilis is responsible for myo-inositol catabolism involving multiple and stepwise reactions. Previous studies demonstrated that IolG and IolE are the enzymes for the first and second reactions, namely dehydrogenation of myo-inositol to give 2-keto-myo-inositol and the subsequent dehydration to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione. In the present studies the third reaction was shown to be the hydrolysis of 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione catalyzed by IolD to yield 5-deoxy-d-glucuronic acid. The fourth reaction was the isomerization of 5-deoxy-D-glucuronic acid by IolB to produce 2-deoxy-5-keto-D-gluconic acid. Next, in the fifth reaction 2-deoxy-5-keto-D-gluconic acid was phosphorylated by IolC kinase to yield 2-deoxy-5-keto-D-gluconic acid 6-phosphate. IolR is known as the repressor controlling transcription of the iol operon. In this reaction 2-deoxy-5-keto-D-gluconic acid 6-phosphate appeared to be the intermediate acting as inducer by antagonizing DNA binding of IolR. Finally, IolJ turned out to be the specific aldolase for the sixth reaction, the cleavage of 2-deoxy-5-keto-D-gluconic acid 6-phosphate into dihydroxyacetone phosphate and malonic semialdehyde. The former is a known glycolytic intermediate, and the latter was previously shown to be converted to acetyl-CoA and CO(2) by a reaction catalyzed by IolA. The net result of the inositol catabolic pathway in B. subtilis is, thus, the conversion of myo-inositol to an equimolar mixture of dihydroxyacetone phosphate, acetyl-CoA, and CO(2).

Genetic modification of Bacillus subtilis for production of D-chiro-inositol, an investigational drug candidate for treatment of type 2 diabetes and polycystic ovary syndrome.

D-chiro-inositol (DCI) is a drug candidate for the treatment of type 2 diabetes and polycystic ovary syndrome, since it improves the efficiency with which the body uses insulin and also promotes ovulation. Here, we report genetic modification of Bacillus subtilis for production of DCI from myo-inositol (MI). The B. subtilis iolABCDEFGHIJ operon encodes enzymes for the multiple steps of the MI catabolic pathway. In the first and second steps, MI is converted to 2-keto-MI (2KMI) by IolG and then to 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione by IolE. In this study, we identified iolI encoding inosose isomerase, which converts 2KMI to 1-keto-D-chiro-inositol (1KDCI), and found that IolG reduces 1KDCI to DCI. Inactivation of iolE in a mutant constitutively expressing the iol operon blocked the MI catabolic pathway to accumulate 2KMI, which was converted to DCI via the activity of IolI and IolG. The mutant was able to convert at least 6% of input MI in the culture medium to DCI.

Identification of a functional 2-keto-myo-inositol dehydratase gene of Sinorhizobium fredii USDA191 required for myo-inositol utilization.

Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His(6)-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE.

Identification of additional TnrA-regulated genes of Bacillus subtilis associated with a TnrA box.

Bacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen-limited growth. In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome-wide screening for TnrA-regulated genes associated with a TnrA box. A combination of DNA microarray hybridization and a genome-wide search for TnrA boxes allowed us to find 36 TnrA-regulated transcription units associated with a putative TnrA box. Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which (nasA and nasBCDEF) possessed a common TnrA box. The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA. The TnrA targets detected in this study were nrgAB, pucJKLM, glnQHMP, nasDEF, oppABCDF, nasA, nasBCDEF and ywrD for positive regulation, and gltAB, pel, ywdIJK, yycCB, yttA, yxkC, ywlFG, yodF and alsT for negative regulation, nrgAB and gltAB being well-studied TnrA targets. It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets. The physiological role of the TnrA regulon is discussed.

Bacillus subtilis LmrA is a repressor of the lmrAB and yxaGH operons: identification of its binding site and functional analysis of lmrB and yxaGH.

The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter. LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified. Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance. LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified. The LmrA regulon was thus determined to comprise lmrAB and yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding.