MiR-200c regulates apoptosis of placental trophoblasts in preeclampsia rats through Wnt/ß-catenin signaling pathway.

OBJECTIVE: To investigate the influence of micro ribonucleic acid 200c (miR-200c) on the apoptosis of placental trophoblasts in a rat model of preeclampsia (PE). MATERIALS AND METHODS: PE model in rats was established for extracting placental trophoblasts. Overexpression or knockdown of miR-200c was achieved by transfection of miR-200c mimics or inhibitor. Flow cytometry was carried out to detect the apoptotic rate of placental trophoblasts. Dual-luciferase reporter gene assay was performed to detect the interaction of miR-200c with WNT1. Western blotting was applied to determine the changes of protein levels in placental trophoblasts. RESULTS: The expression level of miR-200c in placental trophoblasts of PE group was significantly higher than that in control group. The apoptosis rate was (22.45 ± 2.62)%, (6.58 ± 1.28)%, and (9.57 ± 1.35)% in miR-200c mimic group, miR-200c inhibitor group, and control group, respectively, showing statistically significant differences. MiR-200c overexpression downregulated the expression level of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2), but upregulated expression levels of apoptotic proteins Bcl-2-associated X protein (Bax) and active Caspase-3. MiR-200c suppressed WNT1 expression through the interaction with the 3'-untranslated region (3'-UTR) of WNT1. The expressions of WNT1 and ß-catenin were up-regulated after miR-200c overexpression, which was reversed by the Wnt/ß-catenin pathway activator. CONCLUSIONS: MiR-200c is involved in the development and progression of PE through the Wnt/ß-catenin signaling pathway.

Role of cytokines in the gene expression of amyloid beta-protein precursor: identification of a 5'-UTR-binding nuclear factor and its implications in Alzheimer's disease.

One of the major neuropathological characteristics of Alzheimer's disease (AD) is the brain depositions of senile plaques that are mainly composed of toxic amyloid beta-peptide (Abeta), which is generated from a family of Abeta containing precursor proteins (AbetaPP; 695-770 amino acids). The role of cytokines and growth factors has been implicated in the pathogenesis of AD. Our goal is to determine the mode of action of cytokines on the regulation of betaPP gene expression. Here we studied the effect of different cytokines on the activity of 5'-untranslated region (5'-UTR) of betaPP mRNA in human astrocytic cells (U-373). We compared betaPP-5'-UTR activity in the presence of interleukin-1 (IL-1alpha and IL-1beta), transforming growth factor (TGF-beta1) and tumor necrosis factor TNF-alpha1. The astrocytic cells, which were treated separately with these agents, were transfected with either the vector (pSV2CAT) or pSV2UTR-CAT construct containing 90 bp of AbetaPP 5'-UTR +54 to 144 bp). This region was cloned upstream of a reporter chloramphenicol acetyl transferase gene (CAT). Our results indicate that the treatment of pSV2UTR-CAT-transfected cells with either IL-1alpha, IL-1beta, TGF-beta1 or TNF-alpha1 stimulated reporter gene activity in a factor-specific manner. This was consistent with their effects on elevating AbetaPP protein levels. Transfection of the same cells with the pSV2CAT vector lacking 5'-UTR resulted in a reduced reporter gene activity with all treatments studied. DNA-gel shift experiments indicate that the 54/144 region binds to a nuclear protein(s) in a cell type specific manner. These results suggest that 5'-UTR of the AbetaPP gene can respond to the stimulation of different cytokines, which likely regulate AbetaPP transcription and translation via regulatory elements present in the AbetaPP promoter and in 5'-UTR, respectively. The characterization of AbetaPP regulatory elements, including the 5'-UTR, will accelerate the development of novel agents against new targets for AD.

A proximal gene promoter region for the beta-amyloid precursor protein provides a link between development, apoptosis, and Alzheimer's disease.

Abnormalities in regulation of the beta-amyloid precursor protein (APP) gene might be a crucial factor in Alzheimer's disease (AD). Our aim is to study the role of a specific proximal APP promoter element under the apoptotic condition. Our transfection studies with APP promoter deletion constructs indicate that each cell type differently regulates promoter activity. The minimum region that was sufficient to drive basal promoter activity in neuronal PC12 and neuroblastoma SK-N-SH cells was -75/+104 and -47/+104 bp, respectively. In SK-N-SH cells, the -47/+104 construct displayed the highest promoter activity, and the -75/-46 region acted as a negative regulatory element. Results from the gel electrophoretic mobility shift assay (EMSA) indicate that the -75/-46 region binds to a distinct DNA-protein complex with nuclear protein(s) from HeLa, PC12, NIH-3T3, and neuroblastoma cells. EMSA results from HeLa cells, which were stimulated by serum starvation (SR), indicate a significant induction in the signal of the DNA-protein complex from controls. EMSA results from PC12 cells, which were subjected to hypoxia, indicate a significant reduction in the signal. Our results suggest that the -75/-46 region binds to a protein that is upregulated in serum starvation, and downregulated in hypoxia. Because serum starvation contributes to the induction of apoptosis, these results suggest a role of the 30-bp proximal APP promoter element in enhanced apoptotic neuronal cell death.

Does nitric oxide synthase contribute to the pathogenesis of Alzheimer's disease?: effects of beta-amyloid deposition on NOS in transgenic mouse brain with AD pathology.

Oxidative stress is a risk factor for Alzheimer's disease (AD) whose major hallmark includes brain depositions of the amyloid beta peptide (Abeta) derived from the beta-amyloid precursor protein (APP). Our aim was to determine whether or not excessive Abeta deposition would alter nitric oxide synthase (NOS) activity, and thereby affect NOS-mediated superoxide formation. We compared NOS activity in brain extracts between Tg mice (expressing APP Swedish double mutation plus presenilin [PS-1] and nontransgenic [nTg] mice. Five brain regions, including cerebral cortex, hippocampus, cerebellum, and striatum from both nTg and Tg mice showed a detectable level of neuronal (n) NOS activity. Cerebellar extracts from both nTg and Tg mice displayed the highest level of nNOS activity, which was fourfold higher than cortical extracts. Although there was an increase in nNOS activity in Tg brain extracts, this did not attain statistical significance. A similar result was obtained for inducible NOS levels. Our results suggest that excess levels of Abeta failed to both trigger NOS activity and change NOS levels.

Functional characterization of the 5'-regulatory region of the murine apolipoprotein gene.

The apolipoprotein E (APOE) gene causes a major risk factor for the development of Alzheimer's disease (AD). To study the transcription control of the mouse (m) APOE gene, we first tested the promoter activity of a 721-base-pair (bp) 5'-flanking region, which is located 771 bp upstream from the translation initiation codon. We cloned the 721-bp region upstream of the reporter chloramphenicol acetyl transferase (CAT) gene into a promoterless vector (pBLCAT3). The mAPOE promoter and vector DNA were separately transfected in rat glial C6 and neuronal PC12 cell lines. The 721-bp APOE region (from position 329 to 1050) is functionally active in different cell lines tested. The serial deletion analysis indicates that the 266-bp promoter region (from 784 to 1050) has the highest and the 67-bp region (from 983 to 1050) the lowest activity on the reporter gene in neuronal and astrocytic cell lines. These studies suggest that the 147-bp region (from 637 to 784) has a negative regulatory effect on the reporter gene. In the gel shift assay, the 67-bp region binds to a specific transcription factor(s) in PC12 nuclear extracts. Our results suggest that mAPOE can also be expressed in neuronal cells in addition to the astrocytic cells. Characterization of mAPOE promoter is important for the AD drug development discovery and APOE transgenic mice studies.

Regulation of promoter activity of the APP gene by cytokines and growth factors: implications in Alzheimer's disease.

One of the major pathological hallmarks of Alzheimer's disease (AD) is the presence of brain depositions of senile plaques that are primarily composed of potentially toxic amyloid beta-peptide (Abeta), which is generated from a family of Abeta-containing precursor proteins (APP; 695-770 amino acids). The role of inflammatory cytokines and growth factors has been implicated in the pathogenesis of AD. Our goal is to study the effects of these factors on the regulation of APP gene expression. Here we compared APP promoter activity in the presence of different growth factors and cytokines such as brain-derived neurotrophic factor (BDNF), interleukin (IL-1), nerve growth factor (NGF), neurotrofin-3 (NT-3), transforming growth factor (TGF-beta1), and tumor necrosis factor (TNF-alpha1). PC12 neuronal cells, which were treated separately with these agents, were transfected with the construct containing either 190 bp APP proximal promoter region (-46 to 144 bp with respect to the transcription start site [+1]), 94 bp APP 5'-untranslated region (UTR, +50 to 144) or other 5'-UTR-deleted regions. Each construct was cloned upstream of a reporter chloramphenicol acetyl transferase gene (CAT). The treatment of PC12 cells with NGF stimulated reporter activity in all constructs tested. The treatment of cells with BDNF, NT3, TGF-beta1, or TNF-alpha stimulated reporter activity in a promoter/UTR-specific manner. Transfection with the complete -46 to 144 region retained the maximum stimulatory activity for any treatment tested in PC12 cells. These results suggest that the regulatory elements of the APP gene respond to the stimulation of different growth factors, cytokines, and interleukins. This is consistent with the effects of the different growth factors, cytokines, and interleukins on APP message and protein levels.

Phenserine regulates translation of beta -amyloid precursor protein mRNA by a putative interleukin-1 responsive element, a target for drug development.

The reduction in levels of the potentially toxic amyloid-beta peptide (Abeta) has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the expression and processing of the Abeta precursor protein (betaAPP). Earlier reports from our laboratory have shown that a novel cholinesterase inhibitor, phenserine, reduces betaAPP levels in vivo. Herein, we studied the mechanism of phenserine's actions to define the regulatory elements in betaAPP processing. Phenserine treatment resulted in decreased secretion of soluble betaAPP and Abeta into the conditioned media of human neuroblastoma cells without cellular toxicity. The regulation of betaAPP protein expression by phenserine was posttranscriptional as it suppressed betaAPP protein expression without altering betaAPP mRNA levels. However, phenserine's action was neither mediated through classical receptor signaling pathways, involving extracellular signal-regulated kinase or phosphatidylinositol 3-kinase activation, nor was it associated with the anticholinesterase activity of the drug. Furthermore, phenserine reduced expression of a chloramphenicol acetyltransferase reporter fused to the 5'-mRNA leader sequence of betaAPP without altering expression of a control chloramphenicol acetyltransferase reporter. These studies suggest that phenserine reduces Abeta levels by regulating betaAPP translation via the recently described iron regulatory element in the 5'-untranslated region of betaAPP mRNA, which has been shown previously to be up-regulated in the presence of interleukin-1. This study identifies an approach for the regulation of betaAPP expression that can result in a substantial reduction in the level of Abeta.

Analysis of the 5'-flanking region of the beta-amyloid precursor protein gene that contributes to increased promoter activity in differentiated neuronal cells.

To study the transcription control of the beta-amyloid precursor protein (betaAPP) in Alzheimer's disease (AD), we functionally characterized the betaAPP gene promoter in differentiated cells. PC12 cells were first differentiated with nerve growth factor (NGF) and then transient transfection analysis was done with a series of 5'-deletion constructs, that extended as far upstream as -7900 down to +104 base pair (bp) relative to the transcription start site (+1). The truncated regions of the promoter were linked upstream to a reporter gene, chloramphenicol acetyl transferase (CAT). The CAT assay was performed to compare promoter activity of different 5'-flanking and intronic regions of the betaAPP gene. Our results suggest that the longest (-7900/+104) and one of the shortest (-47/+104) regions possessed significantly higher levels of promoter activity than the promoterless vector in NGF-differentiated PC12 cells. A deletion of about 7600 bp region from the -7900 to +104 construct resulted in 50% loss of original promoter activity. A deletion of all but 47 bp from the -7900 to +104 construct resulted in the loss of 66% (and retention of 34%) promoter activity. The region -3416/+104 bp displayed the strongest promoter activity whereas +1/+104 bp showed the least activity among all deletion constructs studied. The upstream region -5529 to -3416 contains a negative regulatory element and -3416 to -1131 contains a positive regulatory element. The very upstream region, -7900 to -3411, lacks independent functional activity. The 5'-UTR region (+1 to +104) showed minimum activity and the -75 to +104 region constitutes the basic promoter element. The first exon or a large part of the first intron (+99 to +6200) did not display any significant promoter activity. Thus, several positive and negative regulatory elements influence the basal level of betaAPP promoter activity in NGF-differentiated PC12 cells. We speculate that any structural alteration(s) due to a specific mutation in these regulatory regions can potentially alter the transcriptional machinery, and that can perhaps affect the level of beta-amyloid protein involved in AD.

Effect of a memory-enhancing drug, AIT-082, on the level of synaptophysin.

Our objective is to study the effect of AIT-082 on the level of synaptophysin in cultured pheochromocytoma (PC12) cells. The drug AIT-082, a unique purine hypoxanthine derivative, is under development for the treatment of Alzheimer's disease (AD). We analyzed synaptophysin protein as an index of synaptic numbers and density and indirectly neuronal transmission. PC12 cells were treated with nerve growth factor (NGF) (50 ng/ml) and/or different doses of AIT-082 (5-50 ng/ml) obtained from NeoTherapeutics, CA. In the western immunoblots of conditioned media and cell lysates, we detected synaptophysin as 36-40 kDa protein bands. When PC12 cells were treated with NGF and samples were analyzed at 24 or 48 hours after treatment, the secretion of synaptophysin was drastically reduced in the conditioned medium. A significant reduction in the intracellular levels of synaptophysin in NGF-treated samples was also noted. By contrast, when PC12 cells were treated with AIT-082, the secretion of synaptophysin was increased in the conditioned medium as compared to the control. There was also a significant increase in the intracellular levels of synaptophysin in AIT-082-treated cultures. NGF treatment resulted in sympathetic neuronal phenotypes in PC12 cells. As it is known that the immunoreactivity of the synaptophysin protein correlates with the density of the synaptic terminal, our results suggest that treatment by AIT-082 could enhance neurotransmitter release at the presynaptic terminal, which may play a role in the improvement of cognition seen in AD subjects.

Promoter activity of the beta-amyloid precursor protein gene is negatively modulated by an upstream regulatory element.

Alzheimer's disease (AD) is characterized by the aggregation of the amyloid beta-peptide (Abeta) which is generated from a larger beta-amyloid precursor protein (betaAPP). An overexpression of the betaAPP gene in certain areas of the AD brain has been suggested to be an important factor in the neuropathology of AD. Here we have further characterized an upstream regulatory element (URE) located between -2257 and -2234 of the human betaAPP promoter. In addition to its location in the promoter, BLAST search reveals that URE is present in several introns of the betaAPP gene and is also detected in many other genes. For functional studies, two promoter regions were cloned upstream of the reporter gene, chloramphenicol acetyl transferase (CAT): (i) phbetaE-B - the plasmid that contains the human (h) promoter region (-2832 to +101) including URE, and (ii) prhbetaE-B - the plasmid that contains the rhesus (rh) promoter region excluding URE as it lacks a 270 bp region of the hbetaAPP promoter (-2435 to -2165). Transient transfection studies indicate that phbetaE-B displayed significantly less CAT-promoter activity than prhbetaE-B in C6, PC12 and SK-N-SH cells. To determine the role of URE in a heterologous promoter, a pbetaURE construct was made by subcloning URE in an enhancerless promoter vector pCATP. The pbetaURE-CAT construct displayed threefold to fourfold less promoter activity than pCATP when different cell lines were transfected with the plasmids. URE interacts with a novel protein(s) as determined by the electrophoretic mobility shift assay (EMSA). Although the core DNA region of URE resembles with the NF-kB element, URE-binding protein is not related to the NF-kB transcription factor. When EMSA was performed with specific competitors in different cell lines, the labeled URE probe was not competed by the oligonucleotides specific for either the AP3, NF-1 or NF-kB transcription factor. The migration of the URE-protein complex was different from the NF-kB-protein complex in the EMSA gel. A distinct URE-specific nuclear factor was also detected in frontal cortex of a normal human brain. These results suggest that the URE region acts as a repressor element, that the URE-binding protein is not related to the known transcription factors tested, and that the protein is present in astrocytic, neuroblastoma, PC12 cells and in the human brain.

Increased phosphorylation of HP1, a heterochromatin-associated protein of Drosophila, is correlated with heterochromatin assembly.

The heterochromatin-associated nonhistone chromosomal protein HP1 exerts dosage-dependent effects on the silencing of genes juxtaposed to pericentric heterochromatin in Drosophila melanogaster. Here, we report that HP1 is multiply phosphorylated in Drosophila tissue, predominantly at serine and threonine residues. Pulse-labeling studies of explanted Drosophila tissues suggest that phosphorylation is relatively rapid and that phosphate is incorporated into existing protein. Maternally synthesized HP1 is underphosphorylated. The appearance of more highly phosphorylated HP1 isoforms at 1.5-2 h of development coincides with the embryonic stage at which cytologically visible heterochromatin appears and HP1 concentrates in heterochromatin. The extent of HP1 phosphorylation is lower in polytene tissue, where heterochromatin is underrepresented. These results are consistent with a role for phosphorylation of HP1 in the assembly and maintenance of heterochromatin in Drosophila.