Regulation Of mRNA Levels Research Articles

Antagonistic Effects of Insulin and TGF-β3 during Chondrogenic Differentiation of Human BMSCs under a Minimal Amount of Factors

Growth factors are crucial regulators of cell differentiation towards tissue and organ development. Insulin and transforming growth factor-β (TGF-β) have been used as the major factors for chondrogenesis in vitro, by activating the AKT and Smad signaling pathways. Previous reports demonstrated that AKT and Smad3 have a direct interaction that results in the inhibition of TGF-β-mediated cellular responses. However, the result of this interaction between AKT and Smad3 during the chondrogenesis of human bone marrow-derived stem/progenitor cells (hBMSCs) is unknown. In this study, we performed functional analyses by inducing hBMSCs into chondrogenesis with insulin, TGF-β3 or in combination, and found that TGF-β3, when applied concomitantly with insulin, significantly decreases an insulin-induced increase in mRNA levels of the master regulator of chondrogenesis, SOX9, as well as the regulators of the 2 major chondrocyte markers, ACAN and COL2A1. Similarly, the insulin/TGF-β3-treated group presented a significant decrease in the deposition of cartilage matrix as detected by safranin O staining of histological sections of hBMSC micromass cultures when compared to the group stimulated with insulin alone. Intracellular analysis revealed that insulin-induced activation of AKT suppressed Smad3 activation in a dose-dependent manner. Accordingly, insulin/TGF-β3 significantly decreased the TGF-β3-induced increase in mRNA levels of the direct downstream factor of TGF-β/Smad3, CCN2/CGTF, compared to the group stimulated with TGF-β3 alone. On the other hand, insulin/TGF-β3 stimulation did not suppress insulin-induced expression of the downstream targets TSC2 and DDIT4/REDD1. In summary, insulin and TGF-β3 have antagonistic effects when applied concomitantly, with a minimal number of factors. The application of an insulin/TGF-β3 combination without further supplementation should be used with caution in the chondrogenic differentiation of hBMSCs.

Effects and Molecular Mechanism of GST-Irisin on Lipolysis and Autocrine Function in 3T3-L1 Adipocytes.

Irisin, which was recently identified as a myokine and an adipokine, transforms white adipose tissue to brown adipose tissue and has increasingly caught the attention of the medical and scientific community. However, the signaling pathway of irisin and the molecular mechanisms responsible for the lipolysis effect remain unclear. In this study, we established an efficient system for the expression and purification of GST-irisin in Escherichia coli. The biological activity of GST-irisin was verified using the cell counting kit-8 assay and by detecting the mRNA expression of uncoupling protein 1. Our data showed that GST-irisin regulates mRNA levels of lipolysis-related genes such as adipose triglyceride lipase and hormone-sensitive lipase and proteins such as the fatty acid-binding protein 4, leading to increased secretion of glycerol and decreased lipid accumulation in 3T3-L1 adipocytes. In addition, exogenous GST-irisin can increase its autocrine function in vitro by regulating the expression of fibronectin type III domain-containing protein 5. GST-irisin could regulate glucose uptake in 3T3-L1 adipocytes. Hence, we believe that recombinant GST-irisin could promote lipolysis and its secretion in vitro and can potentially prevent obesity and related metabolic diseases.

Genetic Variation in Renal Expression of Folate Receptor 1 (Folr1) Gene Predisposes Spontaneously Hypertensive Rats to Metabolic Syndrome.

Metabolism of homocysteine and other sulfur amino acids is closely associated with metabolism of folates. In this study, we analyzed the possible role of folates and sulfur amino acids in the development of features of the metabolic syndrome in the BXH/HXB recombinant inbred strains derived from the spontaneously hypertensive rat (SHR) and Brown Norway progenitors. We mapped a quantitative trait locus for cysteine concentrations to a region of chromosome 1 that contains a cis-acting expression quantitative trait locus regulating mRNA levels of folate receptor 1 (Folr1) in the kidney. Sequence analysis revealed a deletion variant in the Folr1 promoter region of the SHR. Transfection studies demonstrated that the SHR-promoter region of Folr1 is less effective in driving luciferase reporter gene expression than the Brown Norway promoter region of Folr1. Results in the SHR.BN-chr.1 congenic strain confirmed that the SHR variant in Folr1 cosegregates with markedly reduced renal expression of Folr1 and renal folate reabsorption, decreased serum levels of folate, increased serum levels of cysteine and homocysteine, increased adiposity, ectopic fat accumulation in liver and muscle, reduced muscle insulin sensitivity, and increased blood pressure. Transgenic rescue experiments performed by expressing a Folr1 transgene in the SHR ameliorated most of the metabolic disturbances. These findings are consistent with the hypothesis that inherited variation in the expression of Folr1 in the kidney influences the development of the metabolic syndrome and constitutes a previously unrecognized genetic mechanism that may contribute to increased risk for diabetes mellitus and cardiovascular disease.

Deficiency of dietary niacin impaired intestinal mucosal immune function via regulating intestinal NF-κB, Nrf2 and MLCK signaling pathways in young grass carp (Ctenopharyngodon idella).

This study investigated the effects of dietary niacin on intestinal mucosal immune and physical barrier, and relative mRNA levels of signaling molecules in the intestine of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (255.63±0.41g) were fed six diets containing graded levels of niacin (3.95, 14.92, 24.98, 35.03, 44.97 and 55.01mg/kg diet) for 8 weeks. Results observed that niacin deficiency decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 (C3) content in the intestine (P<0.05), down-regulated mRNA levels of liver expressed antimicrobial peptide 2 (LEAP-2), hepcidin, interleukin 10, transforming growth factor β1 and inhibitor of κBα (IκBα) (P<0.05), up-regulated tumor necrosis factor α, interleukin 1β, interferon γ2, interleukin 8, nuclear factor kappa B P65 (NF-κB P65), IκB kinase α (IKKα), IκB kinase β (IKKβ) and IκB kinase γ (IKKγ) in all intestinal segments of young grass carp (P<0.05). In addition, niacin deficiency increased reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) contents, decreased glutathione content, and copper/zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferases (GST) and glutathione reductase (GR) activities in the intestine of young grass carp (P<0.05). Additionally, niacin deficiency decreased mRNA levels of CuZnSOD, MnSOD, GPx, CAT, GST, GR, Claudin b, Claudin 3, Claudin c, Occludin, ZO-1, Claudin 15 and NF-E2-related factor 2 (Nrf2) (P<0.05), and increased Claudin 12, Kelch-like ECH-associating protein 1a (Keap1a), myosin light-chain kinase (MLCK) and p38 mitogen-activated protein kinase (p38 MAPK) mRNA expression levels in the intestine of young grass carp (P<0.05), while the mRNA level of Kelch-like ECH-associating protein 1b (Keap1b) did not change (P>0.05). In conclusion, niacin deficiency decreased intestinal mucosal immune and intestinal physical function, as well as regulated mRNA levels of NF-κB P65, IκBα, IKKα, IKKβ, IKKγ, Nrf2, Keap1a, p38 MAPK and MLCK in the intestine of young grass carp. Based on the broken-line model analysis of intestinal lysozyme activity, the requirement of niacin for young grass carp (255.63±0.41g) were estimated to be 39.80mg/kg diet.

A Dynamic Intron Retention Program in the Mammalian Megakaryocyte and Erythrocyte Lineages

Intron retention (IR), the least studied form of alternative splicing, has recently been shown to have important biological roles in a variety of cell types. While it can alter a gene's protein-coding sequence, it is becoming particularly well-known for its potential to impact gene expression by destabilizing mRNAs through the nonsense-mediated decay pathway or by promoting their retention in the nucleus. A complex, dynamic, and biologically important IR program has been described in maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We therefore globally assessed IR in the mammalian erythroid and megakaryocyte lineages. Intron Retention Finder, a bioinformatics tool that measures IR in RNA-seq datasets, was used to analyze IR in primary cells of the erythroid and megakaryocyte lineages as well as their common progenitor cells. Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes. Complex IR patterns were seen in murine erythropoiesis from the megakaryocytic-erythroid branch point throughout the terminal maturation stages. Within the terminally differentiating proerythroblast to orthochromatic erythroblast stages, hundreds of introns saw their retention level increase as cells differentiate while a smaller set exhibited an opposing trend. Similarly complex patterns including a dramatic IR increase in orthochromatic erythroblasts were observed during human terminal erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and megakaryocytes and their overlapping gene expression patterns, the megakaryocytic IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. This suggests that the dynamic IR patterns are not simply the result of general maturational changes, but rather may arise via lineage-specific mechanisms. Importantly, we observed an inverse relationship between IR and gene expression changes, supporting the hypothesis that IR serves to regulate mRNA levels. Our findings add a new dimension to the megakaryocyte and erythroid transcription programs by expanding the mechanisms of gene control to include this understudied form of alternative splicing. DisclosuresNo relevant conflicts of interest to declare.

Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1.

SummaryIn eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these “decay-promoting” introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation.

MiRNA-27b levels are associated with CYP3A activity invitro and invivo.

Previous in vitro studies have shown that microRNA‐27b (miR‐27b) may regulate mRNA levels of CYP3A4, vitamin D receptor (VDR), and Peroxisome proliferator‐activated receptor α (PPAR α) as well as CYP3A4 protein expression and activity. In vitro studies have also shown that vitamin D may affect the expression of CYP3A4. The primary aim of this pilot study was to investigate the association between miR‐27b and CYP3A expression and activity. The secondary aim was to investigate the association between 25‐hydroxy vitamin D in serum and CYP3A activity. Mi‐RNA‐27b was quantified using real‐time PCR in serum samples (n = 28) and 25‐hydroxyvitamin D was measured and correlated with the levels of the endogenous CYP3A activity marker 4β‐hydroxycholesterol. In addition, the correlation between miR‐27b and CYP3A activity, measured by dextromethorphan N‐demethylation and 6β‐hydroxylation of testosterone and the gene expression of CYP3A4, VDR and PPAR α were assessed in 20 human liver samples. A significant association between circulatory miR‐27b levels and 4β‐hydroxycholesterol ratio was found; P = 0.04, and between hepatic miR‐27b levels and CYP3A activity, measured by dextromethorphan N‐demethylation in human liver (P = 0.04). There was no association between hepatic miR‐27b and mRNA levels of CYP3A4, VDR or PPAR α. There was a significant association between serum 25‐hydroxyvitamin D levels and 4β‐hydroxycholesterol ratio, P = 0.002. In conclusion, this pilot‐study supports the hypothesis that miR‐27b levels as well as 25‐hydroxyvitamin D may affect CYP3A activity in vivo. The results indicate that miR‐27b exerts its inhibitory effect on a translational level rather than affecting mRNA levels.

Prenatal glucocorticoid exposure programs adrenal PNMT expression and adult hypertension.

Prenatal exposure to glucocorticoids (GCs) programs for hypertension later in life. The aim of the current study was to examine the impact of prenatal GC exposure on the postnatal regulation of the gene encoding for phenylethanolamine N-methyltransferase (PNMT), the enzyme involved in the biosynthesis of the catecholamine, epinephrine. PNMT has been linked to hypertension and is elevated in animal models of hypertension. Male offspring of Wistar-Kyoto dams treated with dexamethasone (DEX) developed elevated systolic, diastolic and mean arterial blood pressure compared to saline-treated controls. Plasma epinephrine levels were also elevated in adult rats exposed to DEX in utero. RT-PCR analysis revealed adrenal PNMT mRNA was higher in DEX exposed adult rats. This was associated with increased mRNA levels of transcriptional regulators of the PNMT gene: Egr-1, AP-2, and GR. Western blot analyses showed increased expression of PNMT protein, along with increased Egr-1 and GR in adult rats exposed to DEX in utero. Furthermore, gel mobility shift assays showed increased binding of Egr-1 and GR to DNA. These results suggest that increased PNMT gene expression via altered transcriptional activity is a possible mechanism by which prenatal exposure to elevated levels of GCs may program for hypertension later in life.

Renal stromal miRNAs are required for normal nephrogenesis and glomerular mesangial survival.

MicroRNAs are small noncoding RNAs that post-transcriptionally regulate mRNA levels. While previous studies have demonstrated that miRNAs are indispensable in the nephron progenitor and ureteric bud lineage, little is understood about stromal miRNAs during kidney development. The renal stroma (marked by expression of FoxD1) gives rise to the renal interstitium, a subset of peritubular capillaries, and multiple supportive vascular cell types including pericytes and the glomerular mesangium. In this study, we generated FoxD1GC;Dicerfl/fl transgenic mice that lack miRNA biogenesis in the FoxD1 lineage. Loss of Dicer activity resulted in multifaceted renal anomalies including perturbed nephrogenesis, expansion of nephron progenitors, decreased renin-expressing cells, fewer smooth muscle afferent arterioles, and progressive mesangial cell loss in mature glomeruli. Although the initial lineage specification of FoxD1+ stroma was not perturbed, both the glomerular mesangium and renal interstitium exhibited ectopic apoptosis, which was associated with increased expression of Bcl2l11 (Bim) and p53 effector genes (Bax, Trp53inp1, Jun, Cdkn1a, Mmp2, and Arid3a). Using a combination of high-throughput miRNA profiling of the FoxD1+-derived cells and mRNA profiling of differentially expressed transcripts in FoxD1GC;Dicerfl/fl kidneys, at least 72 miRNA:mRNA target interactions were identified to be suppressive of the apoptotic program. Together, the results support an indispensable role for stromal miRNAs in the regulation of apoptosis during kidney development.

A synthetic cryptochrome inhibitor induces anti-proliferative effects and increases chemosensitivity in human breast cancer cells

Disruption of circadian rhythm is a major cause of breast cancer in humans. Cryptochrome (CRY), a circadian transcription factor, is a risk factor for initiation of breast cancer, and it is differentially expressed between normal and breast cancer tissues. Here, we evaluated the anti-proliferative and pro-apoptotic activity of KS15, a recently discovered small-molecule inhibitor of CRY, in human breast cancer cells. First, we investigated whether KS15 treatment could promote E-box-mediated transcription by inhibiting the activity of CRY in MCF-7 human breast cancer cells. Protein and mRNA levels of regulators of cell cycle and apoptosis, as well as core clock genes, were differentially modulated in response to KS15. Next, we investigated whether KS15 could inhibit proliferation and increase sensitivity to anti-tumor drugs in MCF-7 cells. We found that KS15 decreased the speed of cell growth and increased the chemosensitivity of MCF-7 cells to doxorubicin and tamoxifen, but had no effect on MCF-10A cells. These findings suggested that pharmacological inhibition of CRY by KS15 exerts an anti-proliferative effect and increases sensitivity to anti-tumor drugs in a specific type of breast cancer.

Dietary pantothenic acid deficiency and excess depress the growth, intestinal mucosal immune and physical functions by regulating NF-κB, TOR, Nrf2 and MLCK signaling pathways in grass carp (Ctenopharyngodon idella)

This study investigated the effects of dietary pantothenic acid (PA) on the growth, intestinal mucosal immune and physical barrier, and relative mRNA levels of signaling molecules in the intestine of grass carp (Ctenopharyngodon idella). A total of 540 grass carp (253.44 ± 0.69 g) were fed six diets with graded levels of PA (PA1, PA15, PA30, PA45, PA60 and PA75 diets) for 8 weeks. The results indicated that compared with PA deficiency (PA1 diet) and excess (PA75 diet) groups, optimal PA supplementation increased (P < 0.05): (1) percent weight gain (PWG), feed intake and feed efficiency; (2) lysozyme activity, complement 3 content, liver-expressed antimicrobial peptide 2 and hepcidin, interleukin 10, transforming growth factor β1 and inhibitor of κBα mRNA levels in some intestinal segments; (3) activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferases and glutathione reductase, and NF-E2-related factor 2 (Nrf2) mRNA level in the whole intestine; (4) Claudin b, Claudin 3, Claudin c, Occludin and ZO-1 mRNA levels in some intestinal segments of grass carp. Conversely, optimal PA supplementation decreased (P < 0.05): (1) tumor necrosis factor α, interleukin 1β, interferon γ2, interleukin 8, nuclear factor κB P65 (NF-κB P65), IκB kinase α, IκB kinase β, IκB kinase γ and target of rapamycin (TOR) mRNA expression levels in some intestinal segments; (2) reactive oxygen species, malondialdehyde and protein carbonyl contents, and Kelch-like ECH-associating protein 1a, Kelch-like ECH-associating protein 1b in the intestine; (3) Claudin 12, Claudin 15a and myosin light-chain kinase (MLCK) mRNA levels in some intestinal segments of grass carp. In conclusion, optimum PA promoted growth, intestinal mucosal immune and physical function, as well as regulated mRNA levels of signaling molecules NF-κB P65, TOR, Nrf2 and MLCK in grass carp intestine. Based on the quadratic regression analysis of PWG and intestinal lysozyme activity, the optimal PA levels in grass carp (253.44–745.25 g) were estimated to be 37.73 mg/kg and 41.38 mg/kg diet, respectively.

Polyphenol-rich blackcurrant extract exerts hypocholesterolaemic and hypoglycaemic effects in mice fed a diet containing high fat and cholesterol.

Obesity is associated with an increased risk of metabolic abnormalities, such as hyperlipidaemia and hyperglycaemia. We investigated whether polyphenol-rich blackcurrant extract (BCE) can prevent high fat/high cholesterol (HF/HC) diet-induced metabolic disturbances in mice. Male C57BL/6J mice were fed a modified AIN-93M diet containing HF/HC (16% fat, 0·25% cholesterol, w/w) or the same diet supplemented with 0·1% BCE (w/w) for 12 weeks. There were no differences in total body weight and liver weight between groups. Plasma total cholesterol (TC) and glucose levels were significantly lower in BCE group than in controls, while plasma TAG levels were not significantly different. There was a decreasing trend in hepatic TAG levels, and histological evaluation of steatosis grade was markedly lower in the livers of mice fed BCE. Although the mRNA levels of major regulators of hepatic cholesterol metabolism, i.e. 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) and LDL receptor (LDLR), were not significantly altered by BCE supplementation, protein expression of mature sterol-regulatory element-binding protein and LDLR was significantly increased with no change in HMGR protein. The expression of proprotein convertase subtilisin/kexin type 9 that facilitates LDLR protein degradation, as well as one of its transcriptional regulators, i.e. hepatocyte nuclear factor 4α, was significantly decreased in the livers of mice fed BCE. Taken together, BCE supplementation decreased plasma TC and glucose, and inhibited liver steatosis, suggesting that this berry may be consumed to prevent metabolic dysfunctions induced by diets high in fat and cholesterol.

Prolonged glutamate excitotoxicity increases GluR1 immunoreactivity but decreases mRNA of GluR1 and associated regulatory proteins indissociated rat retinae invitro.

Glutamate excitotoxicity contributes to damage following injury to the central nervous system via mechanisms including changes in the expression of receptors, calcium overload, oxidative stress and cell death. Excitotoxicity is triggered by glutamate binding to receptors, including calcium permeable AMPA receptors, which can be upregulated under injury conditions. However, the transcriptional response of AMPA receptor regulatory proteins to excitotoxic conditions is unknown. Here, we use real-time quantitative PCR (RT-qPCR), to determine the effect of prolonged glutamate excitotoxicity on the expression of mRNA encoding for GluR1 and AMPA receptor regulatory proteins in dissociated rat retinal cultures that include neuronal (retinal ganglion cell (RGCs)) and glial (Müller) cell populations. mRNA levels of GluR1 and regulators of the GluR1 subunit of AMPA receptors, including Sap97, Cnih2 and Cnih3, decreased following 6, 24 and 48h incubation with 5mM glutamate: related regulators not associated with GluR1 were unaffected. In contrast, GluR1 protein, assessed immunohistochemically, was increased in both RGCs and Müller cells after 24h glutamate exposure: western blotting analysis was inconclusive. Reductions in mRNA of GluR1 and associated regulators occurred prior to cell death, which was first detected at 24h, and substantial by 48h. Exposure to glutamate acutely increased the intracellular calcium concentration in the cultures and by 24h, reactive oxygen species were increased. Our data suggest a negative feedback mechanism in retinal cells, that down-regulates transcription of genes encoding GluR1 regulatory proteins in response to glutamate exposure. Despite this mechanism, GluR1 protein levels remain increased, and are associated with increased reactive species and cell death. Therapeutic strategies targeting calcium permeable AMPA receptors should take into account that increases in GluR1 protein are not necessarily associated with increases in associated mRNA levels over time.

Role of Sex Hormones in the Development and Progression of Hepatitis B Virus-Associated Hepatocellular Carcinoma.

Infection with hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC) in developed countries. Epidemiological reports indicate that the incidence of HBV-related HCC is higher in males and postmenopausal females than other females. Increasing evidence suggests that sex hormones such as androgens and estrogens play an important role in the progression of an HBV infection and in the development of HBV-related HCC. While androgen is supposed to stimulate the androgen signaling pathway and cooperate to the increased transcription and replication of HBV genes, estrogen may play a protecting role against the progression of HBV infections and in the development of HBV-related HCC through decreasing HBV RNA transcription and inflammatory cytokines levels. Additionally, sex hormones can also affect HBV-related hepatocarcinogenesis by inducing epigenetic changes such as the regulation of mRNA levels by microRNAs (miRNAs), DNA methylation, and histone modification in liver tissue. This review describes the molecular mechanisms underlying the gender disparity in HBV-related HCC with the aim of improving the understanding of key factors underneath the sex disparity often observed in HBV infections. Furthermore, the review will propose more effective prevention strategies and treatments of HBV-derived diseases.

Expression of CD44s and CD44v3 by Proximal Tubules Influences the Renal Inflammatory Milieu-Induced by LPS Injection

Systemic inflammatory response syndrome (SIRS) typically causes multiple-organ dysfunction/failure, including acute kidney injury (AKI). CD44 comprises a family of 85–200 kDa transmembrane glycoproteins that are widely expressed by multiple cell types and involved in a variety of inflammatory diseases. The multiple functions of CD44 have been attributed to the existence of numerous CD44 isoforms generated by alternative mRNA splicing as well as by extensive post-translational modifications. Renal CD44 expression is minimal in healthy adult kidneys, whereas in inflammatory renal disorders CD44 expression is markedly induced particularly in injured tubular epithelial cells (TEC) both in human diseases and in animal models. The function of CD44 on TEC remains unclear; previously we showed that the shortest isoform CD44 standard (CD44s) and the long CD44 variant 3-10 (CD44v3) exert opposite effects in fibrotic settings. To assess the contribution of tubular expression of CD44s and CD44v3 in SIRS-associated AKI, we used WT and unique transgenic mice expressing CD44s or CD44v3 specifically on proximal TEC. Mice were subjected to intraperitoneal injection of LPS and were sacrificed 4 and 24 hours later. The presence of CD44-isoforms in TEC did not alter the onset of kidney dysfunction or lymphocyte influx but affected the induction of KIM-1 and pro-/anti-inflammatory cytokines after LPS injection. Transgenic kidneys expressing CD44s/CD44v3 displayed more KIM-1 expression, less TNF-α and IL-1β at 4 hours compared to WT kidneys. At 24 hours, CD44v3-expressing kidneys showed elevated IL-10 and TLR4 negative regulator mRNA levels. Proximal TEC-CD44 influenced the renal inflammatory milieu and TEC-CD44v3 associated with expression of antiinflammatory molecules.

SC-32 * TARGETING PROLIFERATION AND INVASION IN GLIOBLASTOMA VIA MITOTIC KINESINS

The nearly universal tumor recurrence seen in glioblastoma is driven by a myriad of factors including the invasive nature of the disease and a cellular heterogeneity that includes a more treatment resistant population termed glioblastoma stem cells (GSCs). In an effort to identify novel therapeutic targets for GBM with potential efficacy against the GSC sub-population, we queried the mRNA levels of mitotic regulators between GSCs and the neoplastic cells without stem cell characteristic, or non-GSCs. One target, the mitotic kinesin, Eg5, was consistently elevated in GSCs on both the mRNA and protein levels with increased expression correlated with higher-grade glioma and poor prognosis. Highly specific inhibitors of Eg5 are available and several have moved to phase I and II clinical trials in patients with solid malignancies. We found that GSCs exhibited a greater sensitivity to the Eg5 inhibitor ispinesib compared to non-GSCs from the same tumor and that inhibition of Eg5 compromised the key stem cell characteristics of self-renewal and tumor initiation. Using an orthotopic xenograft model, we demonstrated the first in vivo evidence for efficacy of Eg5 inhibition on glioblastoma tumor progression. An unexpected impact on invasion was observed following Eg5 inhibition resulting in the identification of a hitherto unappreciated role for Eg5 outside of cellular proliferation. We therefore propose that Eg5 inhibitors hold promise in preventing treatment resistance and recurrence in GBM and may provide a novel therapeutic target for GSCs.

Short and long-term impact of lipectomy on expression profile of hepatic anabolic genes in rats: a high fat and high cholesterol diet-induced obese model.

ObjectiveTo understand the molecular basis of the short and long-term effects of an immediate shortage of energy storage caused by lipectomy on expression profile of genes involved in lipid and carbohydrate metabolism in high fat and high cholesterol diet-induced obese rats.MethodsThe hepatic mRNA levels of enzymes, regulator and transcription factors involved in glucose and lipid metabolism were analyzed by quantitative real time polymerase chain reaction (RT-qPCR) ten days and eight weeks after lipectomy in obese rats. Body and liver weights and serum biochemical parameters, adiponectin, leptin and insulin were determined.ResultsNo significant difference was observed on the food intake between the lipectomized and sham-operated groups during the experimental period. Ten days after the operation, the lipectomized animals showed significant higher triacylglycerol, glucose and insulin levels, a lower adiponectin concentration than the sham-operated rats, along with significant higher hepatic mRNA levels of hepatocyte nuclear factor 4α (HNF4α) and the enzymes involved in lipogenesis, sterol biosynthesis and gluconeogenesis. The results of immunohistochemical (IHC) analysis also confirmed increased levels of lipogenic enzymes in the liver of lipectomized versus sham-operated animals. The lipectomized group had a significantly lower adiponectin/leptin ratio that was positively correlated to the level of LDL (r = 0.823, P<0.05) and negatively to glucose and insulin (r = −0.821 and −0.892 respectively, P<0.05). Eight weeks after the operation, the lipectomized animals revealed significant higher body and liver weights, weight gain, liver to body weight ratio, hepatic triacylglycerol and serum insulin level.ConclusionsIn response to lipectomy a short term enhancement of the expression of hepatic anabolic genes involved in lipid and carbohydrate metabolism was triggered that might eventually lead to the final extra weight gain. These metabolic changes could be the results of reduced circulating adiponectin that further influences the functions of insulin and hepatic HNF4α.

Adenoviral-mediated placental gene transfer of igf-1 corrects placental insufficiency via restoring placental endothelial progenitor cells

Neuropeptide Y (NPY) has been established as an important regulator of food intake in mammals. However, the involvement of NPY in the feeding behavior of fish has not yet been fully understood. The present study investigated the role of NPY in food intake in grass carp. The full-length cDNA sequence of gc-NPY was 797 bp, and an ORF of 96 amino acids, including the 28-residue signal peptide and 36-residue mature peptide, which had high identities to the NPY of cyprinid fishes. Two receptors Y1 and Y5 which mediate the appetite-stimulating effects of NPY in mammals lost from some of the euteleost genomes. However, sequences of other NPY receptors Y8a and Y8b were obtained from the ESTs library of grass carp in our previous studies. NPY and both Y8 receptors were predominantly expressed in the brain. Cumulative food intake was significantly increased by intracerebroventricular administration of NPY during a 72-h observation period. For better understanding of NPY's action in grass carp, the mRNA levels of appetite regulators, energy metabolism genes and key digestive enzymes were assessed. The results showed that the mRNA levels of Y8b were significantly increased, while the expressions of cholecystokinin (CCK), cocaine and amphetamine regulated transcript (CART), pituitary adenylate cyclase-activating peptide (PACAP), leptin, leptin receptor (leptinR) and leptin receptor overlapping transcript protein (LEPROT) were significantly decreased after NPY injection. In addition, the marked stimulation of insulin-like growth factor1 (IGF1), insulin-like growth factor 1 receptor (IGF1R), lipoprotein lipase (LPL), Stearoyl-CoA desaturase 1 (SCD1), amylase and trypsin mRNA levels in liver were caused by NPY administration, and the mRNA level of uncoupling protein 1(UCP 1) tended to decrease but not significantly. These results indicate that NPY acts as an orexigenic factor in the grass carp, and Y8b may involve in the feeding regulation. NPY not only affects food intake, but also has marked effects on energy metabolism in grass carp. Moreover, NPY seems to closely interact with other appetite regulators, and the present results provide novel insight into these complex interactions. Further understandings of appetite regulation in fish ultimately have the potential to yield methods for improving feed efficiency in aquaculture.

Role of Glucocorticoid-induced Leucine Zipper (GILZ) in Bone Acquisition

Glucocorticoids (GCs) have both anabolic and catabolic effects on bone. However, no GC anabolic effect mediator has been identified to date. Here we show that targeted expression of glucocorticoid-induced leucine zipper (GILZ), a GC anti-inflammatory effect mediator, enhances bone acquisition in mice. Transgenic mice, in which the expression of GILZ is under the control of a 3.6-kb rat type I collagen promoter, exhibited a high bone mass phenotype with significantly increased bone formation rate and osteoblast numbers. The increased osteoblast activity correlates with enhanced osteogenic differentiation and decreased adipogenic differentiation of bone marrow stromal cell cultures in vitro. In line with these changes, the mRNA levels of key osteogenic regulators (Runx2 and Osx) increased, and the level of adipogenic regulator peroxisome proliferator-activated receptor (PPAR) γ2 decreased significantly. We also found that GILZ physically interacts with C/EBPs and disrupts C/EBP-mediated PPARγ gene transcription. In conclusion, our results showed that GILZ is capable of increasing bone acquisition in vivo, and this action is mediated via a mechanism involving the inhibition of PPARγ gene transcription and shifting of bone marrow MSC/progenitor cell lineage commitment in favor of the osteoblast pathway.

Investigation of the hepatotoxicity of flutamide: Pro-survival/apoptotic and necrotic switch in primary rat hepatocytes characterized by metabolic and transcriptomic profiles in microfluidic liver biochips

We investigated the effects of the liver damage induced by flutamide in primary rat hepatocytes using liver microfluidic biochips. Flutamide is a non-steroidal anti-androgenic drug. Two flutamide concentrations, 10μM and 100μM, were used to expose the hepatocytes for 24h under perfusion. Thanks to the maintenance of hepatocyte differentiation phenotype and to the biotransformation performance in the microfluidic cultures, the metabolic ratio analysis of hydroxyflutamide, flutamide-gluthatione and hydroxyflutamide-gluthatione productions demonstrated saturation of the drug’s biotransformation process and the maintenance of a high level of flutamide at 100μM when compared to 10μM. A microarray analysis comparing flutamide (10 or 100μM) with controls revealed a common response for both concentrations illustrated by modulating the expression of the mRNA of genes associated with mitochondrial perturbation, of the proliferator-activated receptors (Ppar) signaling, lipid and fatty acid metabolism, antioxidant defense, and cell death pathways, consistently with in vitro and in vivo reports. Additionally to literature reports, our integration of the transcriptomic profiles demonstrated a specific dose dependent response. We found at 10μM a typical pro-survival/apoptosis network activation (through IGF/PDGFD upstream route and via a downstream up regulation in CREB5, BCL2, IKBKG routes in the PI3K/signaling). We also found a down regulation of mRNA levels in sugar and amino acid metabolism pathways. At 100μM a typical necrosis switch was observed associated with a down regulation of the tight junctions’ pathway, a cellular aggregation and a reduction of the cell viability. Altogether our data demonstrated the potential and the sensitivity of our liver microfluidic cultures to evaluate xenobiotic toxicity by improving in vitro analysis and reproducing both in vitro and in vivo results. Finally, we proposed two integrated synthetic networks to describe the response of rat hepatocytes to both exposure concentrations of flutamide.