YWHAZ Research Articles

Ywhaz/14-3-3ζ Deletion Improves Glucose Tolerance Through a GLP-1-Dependent Mechanism.

Multiple signaling pathways mediate the actions of metabolic hormones to control glucose homeostasis, but the proteins that coordinate such networks are poorly understood. We previously identified the molecular scaffold protein, 14-3-3ζ, as a critical regulator of in vitro β-cell survival and adipogenesis, but its metabolic roles in glucose homeostasis have not been studied in depth. Herein, we report that Ywhaz gene knockout mice (14-3-3ζKO) exhibited elevated fasting insulin levels while maintaining normal β-cell responsiveness to glucose when compared with wild-type littermate controls. In contrast with our observations after an ip glucose bolus, glucose tolerance was significantly improved in 14-3-3ζKO mice after an oral glucose gavage. This improvement in glucose tolerance was associated with significantly elevated fasting glucagon-like peptide-1 (GLP-1) levels. 14-3-3ζ knockdown in GLUTag L cells elevated GLP-1 synthesis and increased GLP-1 release. Systemic inhibition of the GLP-1 receptor attenuated the improvement in oral glucose tolerance that was seen in 14-3-3ζKO mice. When taken together these findings demonstrate novel roles of 14-3-3ζ in the regulation of glucose homeostasis and suggest that modulating 14-3-3ζ levels in intestinal L cells may have beneficial metabolic effects through GLP-1-dependent mechanisms.

Identification of suitable reference genes in the mouse placenta

IntroductionQuantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a reliable tool to analyse gene expression profiles. The expression of housekeeping genes generally serves as a reference for mRNA amount, assuming that it remains stable under pathophysiological and experimental conditions. To date, an empirical validation of reference genes suitable for RT-qPCR-based studies in the mouse placenta is missing. MethodsWe used NormFinder and BestKeeper statistical software to analyse the expression stability of candidate housekeeping genes quantified by RT-qPCR in mouse placentas. ResultsFifteen of 32 potential candidate housekeeping genes analysed on gestation day (gd) 16.5 in mouse placentas exhibited an optimal cycle threshold (Ct). Among them B2m, Polr2a, Ubc, and Ywhaz genes showed the highest expression stability in placentas from control, but also experimentally-challenged mice. These genes as well as the currently widely used housekeeping genes Hprt1, Actb, and Gapdh were selected for further quality assessments. We quantified the Ct values of these selected genes in placental samples obtained from wild-type or genetically engineered dams at different gds, or upon selected experimental interventions known to affect placental phenotype. Among all housekeeping genes analysed, Polr2a was the most stably expressed and its expression stability excelled in combination with Ubc. DiscussionPolr2a, especially in combination with Ubc, can be proposed as highly suitable endogenous reference for gene expression analysis in mouse-derived placental tissue. Moreover, the validation of both genes as a stable reference gene in human placenta-derived tissue strengthens the translational relevance of RT-qPCR findings using mouse placenta.

Altered expression of microRNA-451 in eutopic endometrium of baboons (Papio anubis) with endometriosis.

Are microRNAs (miRs) altered in the eutopic endometrium (EuE) of baboons following the induction of endometriosis? Induction of endometriosis causes significant changes in the expression of eight miRs, including miR-451, in the baboon endometrium as early as 3 months following induction of the disease. Endometriosis is one of the most common gynecological disorders and causes chronic pelvic pain and infertility in women of reproductive age. Altered expression of miRs has been reported in women and has been suggested to play an important role in the pathophysiology of several gynecological disorders including endometriosis. EuE was obtained from the same group of baboons before and 3 months after the induction of endometriosis. The altered expression of miR-451 was validated in the eutopic and ectopic endometrium of additional baboons between 3 and 15 months following disease induction. Timed endometrial biopsies from women with and without endometriosis were also used to validate the expression of miR-451. Total RNA was extracted from EuE samples before and after the induction of endometriosis, and miRNA expression was analyzed using a 8 × 15 K miR microarray. Microarray signal data were preprocessed by AgiMiRna software, and an empirical Bayes model was used to estimate the changes. The present study focused on quantitative RT-PCR validation of the microarray data, specifically on miR-451 and its target genes in both baboons (n = 3) and women [control (n = 7) and endometriosis (n = 19)]. Descriptive and correlative analysis of miR-451 and target gene expression was conducted using in situ hybridization and immunohistochemistry, while functional analysis utilized an in vitro 3' untranslated region (UTR) luciferase assay and overexpression of miR-451 in human endometrial and endometriotic cell lines. Induction of endometriosis results in the altered expression of miR-451, -141, -29c, -21, -424, -19b, -200a and -181a in the baboon endometrium. In the baboon, induction of endometriosis significantly decreased the expression of miR-451 at 3 months (P < 0.001), which was also associated with increased expression of its target gene YWHAZ (14.3.3ζ). A similar significant (P < 0.0001) decrease in miR-451 expression was observed in women with endometriosis. The 3' UTR luciferase assay confirmed the regulation of YWHAZ expression by miR-451. Furthermore, overexpression of miR-451 in 12Z cells (immortalized human endometriotic epithelial cell line) led to the decreased expression of its target YWHAZ and this was correlated with decreased cell proliferation. The study focused only on miR-451 and one of its targets, namely YWHAZ. A single miR could target number of genes and a single gene could also be regulated by number of miRs; hence, it is possible that other miRs and their regulated genes may contribute to the pathophysiology of endometriosis. Our data suggest that the presence of ectopic lesions in baboon causes changes in EuE miR expression as early as 3 months postinduction of the disease, and some of these changes may persist throughout the course of the disease. We propose that the marked down-regulation of miR-451 in both baboons and women with endometriosis increases the expression of multiple target genes. Increased expression of one of the target genes, YWHAZ, increases proliferation, likely contributing to the pathophysiology of the disease.

Identification of stably expressed reference genes for RT-qPCR data normalization in defined localizations of cyclic bovine ovaries.

Ovaries are highly complex organs displaying morphological, molecular and functional differences between their cortical zona parenchymatosa and medullary zona vasculosa, and also between the different cyclic luteal stages. Objective of the present study was to validate expression stability of twelve putative reference genes (RGs) in bovine ovaries, considering the intrinsic heterogeneity of bovine ovarian tissue with regard to different luteal stages and intra-ovarian localizations. The focus was on identifying RGs, which are suitable to normalize RT-qPCR results of ovaries collected from clinical healthy cattle, irrespective of localization and the hormonal stage. Expression profiles of twelve potential reference genes (GAPDH, ACTB, YWHAZ, HPRT1, SDHA, UBA52, POLR2C, RPS9, ACTG2, H3F3B, RPS18 and RPL19) were analysed. Evaluation of gene expression differences was performed using genorm, normfinder, and bestkeeper software. The most stably expressed genes according to genorm, normfinder and bestkeeper approaches contained the candidates H3F3B, RPS9, YWHAZ, RPS18, POLR2C and UBA52. Of this group, the genes YWHAZ, H3F3B and RPS9 could be recommended as best-suited RGs for normalization purposes on healthy bovine ovaries irrespective of the luteal stage or intra-ovarian localization.

Involvement of miR-30c in resistance to doxorubicin by regulating YWHAZ in breast cancer cells

MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression implicated in cancer, which play crucial roles in diverse biological processes, such as development, differentiation, apoptosis, and proliferation. The aim of this study was to investigate whether miR-30c mediated the resistance of breast cancer cells to the chemotherapeutic agent doxorubicin (ADR) by targeting tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ). miR-30c was downregulated in the doxorubicin-resistant human breast cancer cell lines MCF-7/ADR and MDA-MB-231/ADR compared with their parental MCF-7 and MDA-MB-231 cell lines, respectively. Furthermore, we observed that transfection of an miR-30c mimic significantly suppressed the ability of MCF-7/ADR to resist doxorubicin. Moreover, the anti-apoptotic gene YWHAZ was confirmed as a target of miR-30c by luciferase reporter assay, and further studies indicated that the mechanism for miR-30c on the sensitivity of breast cancer cells involved YWHAZ and its downstream p38 mitogen-activated protein kinase (p38MAPK) pathway. Together, our findings provided evidence that miR-30c was one of the important miRNAs in doxorubicin resistance by regulating YWHAZ in the breast cancer cell line MCF-7/ADR.

208 COMPARISON OF THE EXPRESSION OF SOX2 AND Stat3 IN BOVINE BLASTOCYST AND HATCHED BLASTOCYST

The investigation of the transcription factors involved in the regulation of pluripotency in pre-implantation embryos of cattle provides important information regarding the early embryonic development and derivation of embryonic stem cells. The present experiment aimed to compare the level of expression of SOX 2 and Stat3, pluripotency markers, in bovine blastocysts (D7) and hatched blastocysts (D10), because it is known that around hatching, the inner cell mass (ICM) differentiates into hypoblast and epiblast. Cumulus–oocyte complexes were matured in TCM 199 for 24 h and fertilized with frozen–thawed sperm. Presumptive zygotes were cultured in SOFaaci for 7 days (group 1) and for 10 days (group 2). All embryos were washed 3 times in PBS, pooled, and frozen at –80°C until RNA extraction. For quantification of SOX2 and Stat3 mRNA levels, total RNA was isolated from pools of 7 embryos per replicate (n = 3) and for each examined developmental stage using RNeasy Micro Kit (Qiagen). The reverse transcriptase Superscript III (Invitrogen) was used for the synthesis of cDNA (cDNA), and the qPCR was performed with the Gotaq qPCR Master Mix (Promega, Madison, WI, USA). As negative control, cDNA was replaced by nuclease-free water in the qPCR reaction. Quantification of expression was determined by the relative standard curve method and normalized to the housekeeping gene YWHAZ. Standard curves for SOX2, Stat 3, and YWHAZ were derived from 10-fold serial dilutions of bovine DNA and gave correlation coefficients greater than 0.99 and efficiencies greater than 94%. The data from 3 replicates were analysed by ANOVA, and it was found that the level of SOX2 abundance is 9.8-fold higher in D7 blastocyst cells compared with D10 blastocyst cells, and the level of Stat 3 transcription is 1.5-fold higher in blastocyst cells than in hatched blastocysts. The expression of pluripotency markers SOX2 and Stat3 was significantly higher in embryos with 7 days of development because at this stage the MCI had not yet begun the process of differentiation. Support of FAPESP is acknowledged.

Genomic Alterations in Breast Cancer Patients in Betel Quid and Non Betel Quid Chewers

Betel Quid (BQ) chewing independently contributes to oral, hepatic and esophageal carcinomas. Strong association of breast cancer risk with BQ chewing in Northeast Indian population has been reported where this habit is prodigal. We investigated genomic alterations in breast cancer patients with and without BQ chewing exposure. Twenty six BQ chewers (BQC) and 17 non BQ chewer (NBQC) breast cancer patients from Northeast India were analyzed for genomic alterations and pathway networks using SNP array and IPA. BQC tumors showed significantly (P<0.01) higher total number of alterations, as compared with NBQC tumors, 48±17% versus 32±25 respectively. Incidence of gain in fragile sites in BQC tumors were significantly (P<0.001) higher as compared with NBQC tumors, 34 versus 23% respectively. Two chromosomal regions (7q33 and 21q22.13) were significantly (p<0.05) associated with BQC tumors while two regions (19p13.3–19p12 and 20q11.22) were significantly associated with NBQC tumors. GO terms oxidoreductase and aldo-keto reductase activity in BQC tumors in contrast to G-protein coupled receptor protein signaling pathway and cell surface receptor linked signal transduction in NBQC tumors were enriched in DAVID. One network “Drug Metabolism, Molecular Transport, Nucleic Acid Metabolism” including genes AKR1B1, AKR1B10, ETS2 etc in BQC and two networks “Molecular Transport, Nucleic Acid Metabolism, Small Molecule Biochemistry” and “Cellular Development, Embryonic Development, Organismal Development” including genes RPN2, EMR3, VAV1, NNAT and MUC16 etc were seen in NBQC. Common alterations (>30%) were seen in 27 regions. Three networks were significant in common regions with key roles of PTK2, RPN2, EMR3, VAV1, NNAT, MUC16, MYC and YWHAZ genes. These data show that breast cancer arising by environmental carcinogens exemplifies genetic alterations differing from those observed in the non exposed ones. A number of genetic changes are shared in both tumor groups considered as crucial in breast cancer progression.

Folate transporter expression decreases in the human placenta throughout pregnancy and in pre-eclampsia

The transport of folate across the placenta involves a number of different receptors including folate receptor-alpha (FR-α), reduced folate carrier (RFC) and proton coupled folate transporter (PCFT). In addition there are a number of ATP-dependent transporters which have also recently been shown to be involved in folate transport; these include ABCB1, ABCC2 and BCRP (ABCG2). The aim of the current study was to characterise the placental mRNA and protein expression of these folate transporters throughout gestation and also to see if expression is altered in pre-eclampsia. Placental tissue was collected from women undergoing termination of pregnancy (TOP) and from women undergoing elective Caesarean section. To investigate mRNA expression quantitative real time PCR was used with gene specific oligonucleotide primers to FR-α, RFC, PCFT, ABCB1, ABCC2, BCRP and the reference gene YWHAZ. Protein expression was also characterised using immunohistochemistry of paraffin embedded placental tissue. Both protein and mRNA expression of all transporters examined decreased as the gestation progressed. Expression of FR-α and PCFT mRNA and protein were decreased in pre-eclampsia compared with normal term pregnancy. The higher levels of expression of FR-α, RFC, PCFT, ABCB1, ABCC2 and BCRP in early pregnancy indicate that these transporters may have an important role in the establishment and development of the placenta, with expression reducing in preparation for parturition. Reductions in FR-α and PCFT in pre-eclampsia may be a mechanism involved in the pathogenesis of pre-eclampsia by limiting placental folate uptake resulting in reduced levels of angiogenesis, cell proliferation and antioxidant protection.

Genome-wide associated loci influencing interleukin (IL)-10, IL-1Ra, and IL-6 levels in African Americans

Interleukins (ILs) are key mediators of the immune response and inflammatory process. Plasma levels of IL-10, IL-1Ra, and IL-6 are associated with metabolic conditions, show large inter-individual variations, and are under strong genetic control. Therefore, elucidation of the genetic variants that influence levels of these ILs provides useful insights into mechanisms of immune response and pathogenesis of diseases. We conducted a genome-wide association study (GWAS) of IL-10, IL-1Ra, and IL-6 levels in 707 non-diabetic African Americans using 5,396,780 imputed and directly genotyped single nucleotide polymorphisms (SNPs) with adjustment for gender, age, and body mass index. IL-10 levels showed genome-wide significant associations (p < 5 × 10(-8)) with eight SNPs, the most significant of which was rs5743185 in the PMS1 gene (p = 2.30 × 10(-10)). We tested replication of SNPs that showed genome-wide significance in 425 non-diabetic individuals from West Africa, and successfully replicated rs17365948 in the YWHAZ gene (p = 0.02). IL-1Ra levels showed suggestive associations with two SNPs in the ASB3 gene (p = 2.55 × 10(-7)), ten SNPs in the IL-1 gene family (IL1F5, IL1F8, IL1F10, and IL1Ra, p = 1.04 × 10(-6) to 1.75 × 10(-6)), and 23 SNPs near the IL1A gene (p = 1.22 × 10(-6) to 1.63 × 10(-6)). We also successfully replicated rs4251961 (p = 0.009); this SNP was reported to be associated with IL-1Ra levels in a candidate gene study of Europeans. IL-6 levels showed genome-wide significant association with one SNP (RP11-314E23.1; chr6:133397598; p = 8.63 × 10(-9)). To our knowledge, this is the first GWAS on IL-10, IL-1Ra, and IL-6 levels. Follow-up of these findings may provide valuable insight into the pathobiology of IL actions and dysregulations in inflammation and human diseases.

Evaluation of endogenous control genes for gene expression studies across multiple tissues and in the specific sets of fat- and muscle-type samples of the pig

To normalize a set of quantitative real-time PCR (q-PCR) data, it is essential to determine an optimal number/set of housekeeping genes, as the abundance of housekeeping genes can vary across tissues or cells during different developmental stages, or even under certain environmental conditions. In this study, of the 20 commonly used endogenous control genes, 13, 18 and 17 genes exhibited credible stability in 56 different tissues, 10 types of adipose tissue and five types of muscle tissue, respectively. Our analysis clearly showed that three optimal housekeeping genes are adequate for an accurate normalization, which correlated well with the theoretical optimal number (r ≥ 0.94). In terms of economical and experimental feasibility, we recommend the use of the three most stable housekeeping genes for calculating the normalization factor. Based on our results, the three most stable housekeeping genes in all analysed samples (TOP2B, HSPCB and YWHAZ) are recommended for accurate normalization of q-PCR data. We also suggest that two different sets of housekeeping genes are appropriate for 10 types of adipose tissue (the HSPCB, ALDOA and GAPDH genes) and five types of muscle tissue (the TOP2B, HSPCB and YWHAZ genes), respectively. Our report will serve as a valuable reference for other studies aimed at measuring tissue-specific mRNA abundance in porcine samples.

Amplification of LAPTM4B and YWHAZ contributes to chemotherapy resistance and recurrence of breast cancer.

Post-surgery adjuvant chemotherapy for breast cancer has effectively reduced metastatic recurrence rates1. However, a significant proportion of women suffer recurrent cancer at distant metastatic sites despite adjuvant treatment. Identification of the genes critical for tumor response to specific chemotherapy drugs is a challenge, but necessary to improve outcomes2. Using integrated genomics, we identified a small number of over-expressed and amplified genes from chromosome 8q22 significantly associated with early disease recurrence despite anthracycline-based adjuvant chemotherapy. The association was confirmed in an analysis of multiple independent cohorts. Two of these genes, the anti-apoptotic gene YWHAZ, and LAPTM4B, a novel lysosomal gene, sensitized tumor cells to anthracyclines when either was depleted by siRNA knockdown and induced drug resistance when either was over-expressed. Over-expression of LAPTM4B resulted in sequestration of drug, delaying its appearance in the nucleus. Over-expression of these two genes was associated with poor tumor response to anthracycline treatment in a neo-adjuvant chemotherapy trial in women with primary breast cancer. Our results suggest that 8q22 amplification and over-expression of LAPTM4B and YWHAZ contribute to de novo chemoresistance to anthracyclines, and are permissive for metastatic recurrence. These two genes may predict anthracycline resistance and influence selection of chemotherapy.

Regulation of IL2 and NUCB1 in Mononuclear Cells Treated With Acyl Glucuronide of Mycophenolic Acid Reveals Effects Independent of Inosine Monophosphate Dehydrogenase Inhibition

The aim of the present study was to investigate whether the acyl glucuronide of mycophenolic acid (AcMPAG) directly affects gene expression independent of guanosine (G) depletion. Human native mononuclear cells from healthy volunteers were studied. A concentration of 100 micromol/L (50 mg/L) AcMPAG, which provided effective inhibition of cell proliferation according to dose-response curves, was selected for gene expression analysis on microarray, verified by quantitative real-time polymerase chain reaction on the LightCycler. Differentially regulated genes on the microarray were 114 inosine monophosphate dehydrogenase-independent genes involved in cell proliferation, signal transduction, chemokine stimulation, endocytosis, vesicle transport, cell adhesion, and cytoskeleton. For verification, 16 genes, which were directly or indirectly related to cell proliferation, were selected for quantitative real-time polymerase chain reaction. SCNM1, ANP32E, CXCL13, CALM1, DKFZp451J0118, TPM3, CDC42, YWHAE, CXCL3, RDX, NDUFA3, and SOD1 showed no significant difference between the studied groups (P > 0.05). CCL1 gene expression was significantly regulated (P < 0.05) only in the mononuclear cell group treated with AcMPAG, whereas YWHAZ gene expression was significantly regulated only in the group treated with AcMPAG in presence of G and 8-aminoguanosine. The difference of interleukin 2 (IL2) and nucleobindin 1 (NUCB1) expression was significant between control and AcMPAG (P < 0.05) however not significant between AcMPAG in presence and absence of G and 8-aminoguanosine (P > 0.05). The expression of interleukin 2 and nucleobindin 1 revealed an effect of AcMPAG on gene expression independent of inosine monophosphate dehydrogenase inhibition.

An association study between polymorphisms in three genes of 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein) family and paranoid schizophrenia in northern Chinese population

We performed an association study between three SNPs in the genes of 14-3-3 family and paranoid schizophrenia. SNP rs983583 G/A in the YWHAZ gene showed significant association with paranoid schizophrenia. Our study indicated that the YWHAZ gene was a potential susceptibility gene for paranoid schizophrenia in the population studied.