Pleckstrin Homology-like Domain Family Research Articles

Correction: A new immunohistochemistry prognostic score (IPS) for recurrence and survival in resected pancreatic neuroendocrine tumors (PanNET).

// Antonio Viudez 1, 2 , Filipe L.F. Carvalho 1 , Zahra Maleki 3 , Marianna Zahurak 4 , Daniel Laheru 1 , Alejandro Stark 1 , Nilofer Z. Azad 1 , Christopher L. Wolfgang 1 , Stephen Baylin 1 , James G. Herman 1 , Ana De Jesus-Acosta 1 1 Department of Medical Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Medical Institutions, Baltimore, Maryland, USA 2 Department of Medical Oncology, Complejo Hospitalario de Navarra-Instituto de Investigaciones Sanitarias de Navarra-IDISNA, Pamplona, Navarra, Spain 3 Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland, USA 4 The Division of Biostatistics and Bioinformatics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Correspondence to: Antonio Viudez, e-mail: antoniovb76@gmail.com , antonio.viudez.berral@navarra.es Keywords: pancreatic neuroendocrine tumor, MGMT, NDRG-1, PHLDA-3, immunohistochemistry, Pathology Section Received: October 01, 2015 Accepted: January 23, 2016 Published: February 17, 2016 ABSTRACT Pancreatic neuroendocrine tumor (PanNET) is a neoplastic entity in which few prognostic factors are well-known. Here, we aimed to evaluate the prognostic significance of N-myc downstream-regulated gen-1 (NDRG-1), O6-methylguanine DNA methyltransferase (MGMT) and Pleckstrin homology-like domain family A member 3 (PHLDA-3) by immunohistochemistry (IHC) and methylation analysis in 92 patients with resected PanNET and follow-up longer than 24 months. In multivariate analyses, ki-67 and our immunohistochemistry prognostic score (IPS-based on MGMT, NDRG-1 and PHLDA-3 IHC expression) were independent prognostic factors for disease-free-survival (DFS), while age and IPS were independent prognostic factors for overall survival (OS). Our IPS could be a useful prognostic biomarker for recurrence and survival in patients following resection for PanNET.

Recognition and characterization of Erythropoietin binding-proteins in the brain of mice.

Erythropoietin (EPO), is a 34KDa glycoprotein hormone, which belongs to type 1 cytokine superfamily. EPO involves in erythrocyte maturation through inhibition of apoptosis in erythroid cells. Besides its main function, protective effects of EPO in heart and brain tissues have been reported. EPO has a critical role in development, growth, and homeostasis of brain. Furthermore EPO has great potential in the recovery of different brain diseases which are still under studying. In this research, EPO binding pattern to brain proteins in animal model was studied. EPO antibody was covalently crosslinked to protein A/G agarose. in order to interact between EPO and its target in brain, about 5µg EPO added to brain homogenates(500ul of 1 mg/ml) and incubate at 4ο C for 30 min. brain tissue lysate were added to agarose beads, After isolation of target proteins(EPO - protein) both one and two-dimensional gel electrophoresis were performed. Proteins were identified utilizing MALDI-TOF/TOF and MASCOT software. This research showed that EPO could physically interact with eightproteins including Tubulin beta, Actin cytoplasmic 2, T-complex protein 1, TPR and ankyrin repeat-containing protein 1, Centromere-associated protein E, Kinesin-like protein KIF7, Growth arrest-specific protein 2 and Pleckstrin homology-like domain family B member 2. Since EPO is a promising therapeutic drug for the treatment of neurological diseases, identified proteins may help us to have a better understanding about the mechanism of protective effects of EPO in the brain. Our data needs to be validated by complementary bioassays.

Maternal prenatal depression is associated with decreased placental expression of the imprinted gene PEG3.

Maternal prenatal stress during pregnancy is associated with fetal growth restriction and adverse neurodevelopmental outcomes, which may be mediated by impaired placental function. Imprinted genes control fetal growth, placental development, adult behaviour (including maternal behaviour) and placental lactogen production. This study examined whether maternal prenatal depression was associated with aberrant placental expression of the imprinted genes paternally expressed gene 3 (PEG3), paternally expressed gene 10 (PEG10), pleckstrin homology-like domain family a member 2 (PHLDA2) and cyclin-dependent kinase inhibitor 1C (CDKN1C), and resulting impaired placental human placental lactogen (hPL) expression. A diagnosis of depression during pregnancy was recorded from Manchester cohort participants' medical notes (n = 75). Queen Charlotte's (n = 40) and My Baby and Me study (MBAM) (n = 81) cohort participants completed the Edinburgh Postnatal Depression Scale self-rating psychometric questionnaire. Villous trophoblast tissue samples were analysed for gene expression. In a pilot study, diagnosed depression during pregnancy was associated with a significant reduction in placental PEG3 expression (41%, p = 0.02). In two further independent cohorts, the Queen Charlotte's and MBAM cohorts, placental PEG3 expression was also inversely associated with maternal depression scores, an association that was significant in male but not female placentas. Finally, hPL expression was significantly decreased in women with clinically diagnosed depression (44%, p < 0.05) and in those with high depression scores (31% and 21%, respectively). This study provides the first evidence that maternal prenatal depression is associated with changes in the placental expression of PEG3, co-incident with decreased expression of hPL. This aberrant placental gene expression could provide a possible mechanistic explanation for the co-occurrence of maternal depression, fetal growth restriction, impaired maternal behaviour and poorer offspring outcomes.

A new immunohistochemistry prognostic score (IPS) for recurrence and survival in resected pancreatic neuroendocrine tumors (PanNET).

Pancreatic neuroendocrine tumor (PanNET) is a neoplastic entity in which few prognostic factors are well-known. Here, we aimed to evaluate the prognostic significance of N-myc downstream-regulated gen-1 (NDRG-1), O6-methylguanine DNA methyltransferase (MGMT) and Pleckstrin homology-like domain family A member 3 (PHLDA-3) by immunohistochemistry (IHC) and methylation analysis in 92 patients with resected PanNET and follow-up longer than 24 months. In multivariate analyses, ki-67 and our immunohistochemistry prognostic score (IPS-based on MGMT, NDRG-1 and PHLDA-3 IHC expression) were independent prognostic factors for disease-free-survival (DFS), while age and IPS were independent prognostic factors for overall survival (OS). Our IPS could be a useful prognostic biomarker for recurrence and survival in patients following resection for PanNET.

A new immunohistochemistry prognostic score (IPS) for recurrence and survival in pancreatic neuroendocrine tumors (PanNET).

241 Background: We aimed to evaluate the expression and prognostic significance of N-myc downstream-regulated gen-1 (NDRG-1), O6-methylguanine DNA methyltransferase (MGMT) and Pleckstrin homology-like domain family A member 3 (PHLDA-3) by immunohistochemistry (IHC) and methylation analysis in resected pancreatic neuroendocrine tumors (PanNET). Methods: Ninety-two patients with resected primary PanNET and follow-up &gt; 24 months were included in this study. Nuclear staining for MGMT and PHLDA-3 were scored as 0, 1-5%, 6-50% and ≥ 51%; cytoplasmic NDRG-1 staining was scored based on intensity and pattern from 0 to 2. We then grouped IHC scores for MGMT (absent versus any expression); for NDRG-1 (0 versus 1 versus 2) and for PHLDA-3 ( &lt; 50% versus ≥ 51%). Finally, we developed an immunohistochemistry prognostic score (IPS) based on MGMT, NDRG-1 and PHLDA-3 IHC expression to predict disease free survival (DFS) and overall survival (OS). The discriminatory ability of multivariate models combining the IPS and important clinical variables was assessed with Harrel’s c-index (HCI) and a modification of Harrell’s c-index (mHCI). Results: DFS was significantly worse in patients without any expression of MGMT compared with those with any grade of expression (HR: 2.21; 95%CI: 0.97-5.02; p = 0.013), in patients with moderate or high score for NDRG-1 (p = 0.005), and in those with high-expression for PHLDA-3 (HR: 1.94; 95%CI: 1.05,3.6; p = 0.036). A significant difference in OS was observed based on NDRG-1 score (p = 0.013). In multivariate analyses, ki-67 (HR: 2.45; 95% CI: 1.20-5.01; p = 0.01) and IPS (HR: 2.68; 95% CI: 1.60,4.49; p = 0.00018) were independent prognostic factors for DFS, while age (HR: 7.67; 95% CI: 2.14,27.45; p = 0.0017) and IPS (HR: 2.67; 95% CI: 1.11, 6.41; p = 0.03) were independent prognostic factors for OS. HCI for the multivariate DFS and OS models were 0.796 and 0.788, respectively. Conclusions: Our IPS is a useful prognostic biomarker for recurrence and survival in patients following resection for PanNET. Prospective studies are warranted to validate our findings and determine its role for patients’ selection to neo/adjuvant treatments

PHLDA1 Promotes Lung Contusion by Regulating the Toll-Like Receptor 2 Signaling Pathway

Background/Aims: Lung contusion is a potentially lethal injury. Pleckstrin homology-like domain family A, member-1 (PHLDA1) is known to play crucial roles in cell proliferation and apoptosis. In this study, we investigated the biological role of PHLDA1 in lung contusion. Methods: The expression levels of PHLDA1 and TLR2 were detected by real time PCR and western. The cytokines were determined by ELISA. The inflammatory factors were detected by flow cytometry. The lung injury was determined by HE staining. Results: PHLDA1 gene and protein expression levels were up-regulated in a mouse lung-contusion model, together with increased neutrophil and macrophage contents. Down-regulation of PHLDA1 by interfering RNA (siPHLDA1 mice) decreased lung injury and neutrophil infiltration. Inflammatory factors, including interleukin (IL)-1β, IL-6, mouse homolog of human growth-regulated oncogene-α (KC), tumor necrosis factor-α, CC chemokine ligand (CCL) 2, and CCL12 were also decreased in siPHLDA1 mice. Expression levels of Toll-like receptor 2 (TLR2) were increased in the lung-contusion mouse model, but were decreased when PHLDA1 was down-regulated. Conclusion: These results demonstrate that PHLDA1 plays a critical role in the development of progressive lung contusion and subsequent inflammation. This information furthers our understanding of the pathogenesis of lung contusion, and suggests that PHLDA1 blockade may represent a potential therapeutic strategy for the treatment of this injury.

Regulation of PHLDA1 Expression by JAK2-ERK1/2-STAT3 Signaling Pathway.

Toll-like receptor 2 (TLR2)-mediated signaling cascades and gene regulation are mainly involved in diseases, such as immunity and inflammation. In this study, microarray analysis was performed using bone marrow-derived macrophages (BMDM) and Raw 264.7 cells to identify novel proteins involved in the TLR2-mediated cellular response. We found that pleckstrin homology-like domain family, member 1 (PHLDA1) is a novel gene up-regulated by TLR2 stimulation and determined the unique signaling pathway for its expression. Treatment with TLR2 agonist Pam3 CSK4 increased mRNA, protein, and fluorescence staining of PHLDA1. Induction of PHLDA1 by TLR2 stimulation disappeared from TLR2 KO mice-derived BMDM. Among janus kinase (JAK) family members, JAK2 was involved in TLR2-stimulated PHLDA1 expression. Signal transducer and activator of transcription 3 (STAT3) also participated in PHLDA1 expression downstream of the JAK2. Interestingly, ERK1/2 was an intermediate between JAK2 and STAT3. In silico analysis revealed the presence of highly conserved γ-activated sites within mouse PHLDA1 promoter and confirmed the JAK2-STAT3 pathway is important to Pam3 CSK4 -induced PHLDA1 transcription. These findings suggest that the JAK2-ERK1/2-STAT3 pathway is an important signaling pathway for PHLDA1 expression and that these proteins may play a critical role in eliciting TLR2-mediated immune and inflammatory response.

The expression of the imprinted gene pleckstrin homology-like domain family A member 2 in placental tissues of preeclampsia and its effects on the proliferation, migration and invasion of trophoblast cells JEG-3.

Preeclampsia (PE) is one of the most common hypertensive disorders and is a leading cause of morbidity and mortality for pregnant women and perinatal babies. Additionally, pleckstrin homology-like domain family A member 2 (PHLDA2) is associated with placental dysfunction. However, the effect of PHLDA2 on trophoblast cell proliferation, migration and invasion has not been investigated. In this study, 15 PE patients and 15 normal pregnant women were recruited and clinical characteristics were summarized. Pleckstrin homology-like domain family A member 2 levels in placental tissues were examined using real-time PCR and western blot. Overexpression plasmid and PHLDA2 siRNA was introduced into JEG-3 cells, respectively. Cell proliferation was measured using MTT assay and flow cytometry. Cell migration and invasion capacities were assessed by wound healing and Transwell assays. It was found that PE patients collectively presented proteinuria, elevated systolic blood pressure (SBP) and diastolic blood pressure (DBP), and lower gestational ages and birth weights. Pleckstrin homology-like domain family A member 2 levels in the preeclamptic placenta were significantly upregulated. Pleckstrin homology-like domain family A member 2 overexpression significantly arrested cells in the G0/G1 phase, inhibited cell proliferation and suppressed the migration and invasion of JEG-3 cells. Pleckstrin homology-like domain family A member 2 knockdown significantly blocked the cells in the S phase of the cell cycle. Knockdown of PHLDA2 alleviated the inhibition on the migration and invasion of trophoblast cells JEG-3. These findings illustrate that PHLDA2 may participate in PE pathogenesis and indicate its potential application in the early diagnosis of PE.

Downregulated expression of PHLDA1 protein is associated with a malignant phenotype of cholangiocarcinoma

Cholangiocarcinoma is one of the most aggressive types of malignancy, and is associated with poor patient prognosis. Recent findings suggest that a decrease in pleckstrin homology-like domain family A, member 1 (PHLDA1) expression is significant in the induction of cell migration and tumor invasion. The clinicopathological significance of the expression of PHLDA1, and its potential correlation with the expression of CD133 in cholangiocarcinoma have remained to be elucidated. In the present study, PHLDA1 protein expression was investigated by immunohistochemical analysis of 218 cholangiocarcinoma tissue samples, as well as 30 para-neoplastic and 20 normal bile ducts. The expression status of PHLDA1 and CD133 was determined, and these results were analyzed against the age, gender, tumor location and size, histological grade, clinical stage and overall mean survival time of the patients. The expression of PHLDA1 protein was markedly decreased in 35.3% of cholangiocarcinomas, compared with that of the para-neoplastic and normal cholangiocytes. Carcinomas with loss of expression of PHLDA1 were significantly correlated with the tumor site (P=0.001), histological grade (P=0.020) and clinical stage (P=0.0001), but not with age (P=0.085), gender (P=0.456) or size (P=0.413), respectively. Kaplan-Meier survival analysis indicated that the loss of expression of PHLDA1 was significantly correlated with the overall survival time (Log rank=193.861; P=0.0001). Furthermore, the expression of PHLDA1 was found to be inversely correlated with the expression of CD133 (γ=-0.142; P=0.036). These findings suggested that the decreased expression of PHLDA1 may be significant in the carcinogenesis and progression of cholangiocarcinoma, and may represent a novel adjunct marker of disease prognosis.

PHLDA1 is a negative regulator for TLR2-mediated inflammatory response (IRC5P.623)

Abstract Atherosclerosis is a slow and complex disease that causes cardiovascular complications and stroke. toll like receptor 2 (TLR2)-mediated signaling pathways and gene regulation are mainly involved in the early development and progression of this complex disease. We found that pleckstrin homology-like domain family A member 1 (PHLDA1) is a unique gene upregulated by TLR2 stimulation and determined the detail mechanisms the signaling pathway for expression in macrophages. Treatment with Pam3CSK4, a TLR2 agonist, increased PHLDA1 mRNA and protein expression levels. Gain- and loss-of-function analyses revealed that PHLDA1 modulates foam cell formation by TLR2 stimulation and foam cell formation-related molecule such as MCP-1. In sequence analysis revealed the presence of typical g-activated sequence (STAT3) element in the promoters of mice PHLDA1 and confirmed the STAT3 binding sites. Subsequently, we also confirmed the detailed mechanism of PHLDA1 expression through the JAK2-ERK1/2-STAT3 pathway. These findings suggest that PHLDA1 is a novel negative regulator aimed at TLR2 and may play a vital role eliciting TLR2-mediated responses in atherosclerosis.

PHLDA2 is a key oncogene-induced negative feedback inhibitor of EGFR/ErbB2 signaling via interference with AKT signaling.

Pleckstrin homology-like domain family A member 2 (PHLDA2) is located within the tumor suppressor region of 11p15, and its expression is suppressed in several malignant tumor types. We recently identified PHLDA2 as a robustly induced, novel downstream target of oncogenic EGFR/ErbB2 signaling. In an immunohistochemical study, we find that PHLDA2 protein expression correlates positively with AKT activation in human lung cancers corroborating our data that PHLDA2 is induced upon oncogenic activation and might serve as a biomarker for AKT pathway activation. We show that PHLDA2 overexpression inhibits AKT phosphorylation while decreased PHLDA2 expression increases AKT activity. We further find that PHLDA2 competes with the PH domain of AKT for binding of membrane lipids, thereby directly inhibiting AKT translocation to the cellular membrane and subsequent activation. Indeed, PHLDA2 overexpression suppresses anchorage-independent cell growth and decreased PHLDA2 expression results in increased cell proliferation and reduced sensitivity to targeted agents of EGFR/ErbB2-driven cancers demonstrating functional relevance for this interaction. In summary, our studies demonstrate that PHLDA2 is strongly regulated by EGFR/ErbB2 signaling and inhibits cell proliferation via repressing AKT activation in lung cancers in a negative feedback loop. We highlight a novel action for PHLDA2 as a potential biomarker for AKT pathway activation.

The role and interaction of imprinted genes in human fetal growth.

Identifying the genetic input for fetal growth will help to understand common, serious complications of pregnancy such as fetal growth restriction. Genomic imprinting is an epigenetic process that silences one parental allele, resulting in monoallelic expression. Imprinted genes are important in mammalian fetal growth and development. Evidence has emerged showing that genes that are paternally expressed promote fetal growth, whereas maternally expressed genes suppress growth. We have assessed whether the expression levels of key imprinted genes correlate with fetal growth parameters during pregnancy, either early in gestation, using chorionic villus samples (CVS), or in term placenta. We have found that the expression of paternally expressing insulin-like growth factor 2 (IGF2), its receptor IGF2R, and the IGF2/IGF1R ratio in CVS tissues significantly correlate with crown–rump length and birthweight, whereas term placenta expression shows no correlation. For the maternally expressing pleckstrin homology-like domain family A, member 2 (PHLDA2), there is no correlation early in pregnancy in CVS but a highly significant negative relationship in term placenta. Analysis of the control of imprinted expression of PHLDA2 gave rise to a maternally and compounded grand-maternally controlled genetic effect with a birthweight increase of 93/155 g, respectively, when one copy of the PHLDA2 promoter variant is inherited. Expression of the growth factor receptor-bound protein 10 (GRB10) in term placenta is significantly negatively correlated with head circumference. Analysis of the paternally expressing delta-like 1 homologue (DLK1) shows that the paternal transmission of type 1 diabetes protective G allele of rs941576 single nucleotide polymorphism (SNP) results in significantly reduced birth weight (−132 g). In conclusion, we have found that the expression of key imprinted genes show a strong correlation with fetal growth and that for both genetic and genomics data analyses, it is important not to overlook parent-of-origin effects.

Isolating the role of elevated Phlda2 in asymmetric late fetal growth restriction in mice.

Pleckstrin homology-like domain family A member 2 (PHLDA2) is a maternally expressed imprinted gene whose elevated expression has been linked to fetal growth restriction in a number of human studies. In mice, Phlda2 negatively regulates placental growth and limits the accumulation of placental glycogen. We previously reported that a three-copy transgene spanning the Phlda2 locus drove a fetal growth restriction phenotype late in gestation, suggesting a causative role for PHLDA2 in human growth restriction. However, in this mouse model, Phlda2 was overexpressed by fourfold, alongside overexpression of a second imprinted gene, Slc22a18. Here, we genetically isolate the role of Phlda2 in driving late fetal growth restriction in mice. We furthermore show that this Phlda2-driven growth restriction is asymmetrical, with a relative sparing of the brain, followed by rapid catch-up growth after birth, classic features of placental insufficiency. Strikingly, fetal growth restriction showed strain-specific differences, being apparent on the 129S2/SvHsd (129) genetic background and absent on the C57BL6 (BL6) background. A key difference between these two strains is the placenta. Specifically, BL6 placentae possess a more extensive endocrine compartment and substantially greater stores of placental glycogen. Taken together, these data support a direct role for elevated Phlda2 in limiting fetal growth but also suggest that growth restriction only manifests when there is limited placental reserve. These findings should be taken into account in interpreting the results from human studies.

Abstract 18333: Tdag51 Deficiency Protects Against Aortic Vascular Calcification by Attenuating Smooth Muscle Cell Death and Osteoblastic Trans-Differentiation

Vascular Calcification (VC), a severe complication increasing the risk of cardiovascular (CV) mortality and morbidity in ageing, diabetic and chronic renal failure patients. VC occurs as vascular smooth muscle cells (VSMC) differentiate into osteoblast- like cells or undergoing apoptosis, resulting in arterial stiffness, elevated pulse pressure (PP) and left ventricular hypertrophy. Yet, molecular mechanisms modulating VSMC differentiation and viability are unclear. Our previous findings demonstrate that T-cell death associated gene 51 (TDAG51, pleckstrin homology-like domain family member) deficiency protects against atherosclerotic lesions. This study provides evidence that TDAG51 contributes to VC by regulating osteogenic transcription factor RUNX2 expression and VSMC survival. To study aortic calcification (AC), male C57Bl/6 wild-type (TDAG51+/+) and TDAG51 deficient (TDAG51-/-) mice were injected subcutaneously with Vitamin D3 (VD3, 50,000 IU/kg) or vehicle (0.9% Saline) for 10 days, an established model of VC. AC was assessed by plasma Pi and Ca2+ levels and aortic valve, thoracic and abdominal aorta segments stained with von Kossa. Osteogenic differentiation and TDAG51 levels were assessed by immunoblot, qRT-PCR and immunohistochemistry. TDAG51-/- mice are protected against osteogenic differentiation in aortic artery. Hallmark features of VC including elastinolysis, collagen deposition and RUNX2 expression were significantly reduced in TDAG51-/- mice compared to control. TDAG51-/- mice maintained PP levels. Microarray analysis revealed ENPP1 (inhibitor of VC) was increased 15-fold in cultured TDAG51-/- aortic smc. The results were verified by qRT-PCR and immunoblots showing elevated ENPP1 expression in TDAG51-/- in presence or absence of VD3 compared to control. Also plasma pyrophosphate remained elevated in TDAG51-/- mice in presence of VD3, indicating increased endogenous ENPP1 activity. ENPP1 transient knockdown in TDAG51-/- vsmc lead to VC, elevated RUNX2, alkaline phosphatase activity and apoptosis. Our data demonstrate TDAG51 is a novel and key promoter of VC by regulating RUNX2 and ENPP1 levels in VC. TDAG51 loss confers protection against VC, apoptosis and PP elevation thereby reducing CV injury risk.

TDAG51 deficiency promotes oxidative stress-induced apoptosis through the generation of reactive oxygen species in mouse embryonic fibroblasts

Apoptosis has an important role in maintaining tissue homeostasis in cellular stress responses such as inflammation, endoplasmic reticulum stress, and oxidative stress. T-cell death-associated gene 51 (TDAG51) is a member of the pleckstrin homology-like domain family and was first identified as a pro-apoptotic gene in T-cell receptor-mediated cell death. However, its pro-apoptotic function remains controversial. In this study, we investigated the role of TDAG51 in oxidative stress-induced apoptotic cell death in mouse embryonic fibroblasts (MEFs). TDAG51 expression was highly increased by oxidative stress responses. In response to oxidative stress, the production of intracellular reactive oxygen species was significantly enhanced in TDAG51-deficient MEFs, resulting in the activation of caspase-3. Thus, TDAG51 deficiency promotes apoptotic cell death in MEFs, and these results indicate that TDAG51 has a protective role in oxidative stress-induced cell death in MEFs.

244 EXPLORING PUTATIVE PITX2C-DEPENDENT GENES IN MOUSE ATRIA

Paired-like homeodomain transcription factor 2 (<i>Pitx2</i>) is involved in the embryonic left-right (L-R) patterning of heart and lung. So far, there is no known function for PITX2 in the adult heart. We recently demonstrated that <i>Pitx2c</i> is expressed highly and selectively in left atria of adult mice and humans compared to the right atria (Kirchhof <i>et al</i>., 2011), indicative of a functional role for <i>Pitx2c</i> in the adult left atrium. Furthermore, there is a strong association between polymorphisms close to the <i>PITX2</i> locus (on chromosome 4q25) and atrial fibrillation (AF) (Gudbjartsson <i>et al</i>., 2007; Kaab <i>et al</i>., 2009). We hypothesise that alterations in the L-R atrial expression ratio could promote the development of ectopic stimuli and AF. Gene array analysis previously identified a number of genes differentially regulated between left and right atria in wildtype adult mice (Kahr <i>et al</i>., 2011). Indirect comparison with gene arrays performed in conjunction with these experiments, suggested that <i>Ccl21, Ddit4l and Ppp1r1b</i> were downregulated in the left atria of <i>Pitx2c</i> heterozygous mice, suggestive of downstream Pitx2-dependent expression in the adult left atrium. In this current work we have sought to quantify differential expression levels of Chemokine ligand 21 (<i>Ccl21</i>); DNA damage-inducible transcript 4-like (<i>Ddit4l</i>); Pleckstrin homology-like domain family A, member 1 (<i>Phlda1</i>); Protein phosphatase 1 regulatory subunit 1B (<i>Ppp1r1b</i>); Scavenger receptor class A (<i>Scara5</i>); Troponin I2 type 2 (<i>Tnni2</i>) and Chemokine ligand 14 (<i>Cxcl14</i>) by RT-PCR in the left and right atria of 18 mice (9 <i>Pitx2c</i> heterozygous and 9 litter mate controls) aged between 14-20wks. RT-PCR was performed using SYBR Green and the ΔΔCt Method. All genes were found to be differentially expressed between the left and right atria in both wildtype and <i>Pitx2c</i> heterozygous mice. There was a trend towards differential fold change reduction in the left atria of <i>Pitx2c</i> heterozygous mice compared to wildtype for all candidate genes. The L-R ratios between <i>Ccl21</i> and <i>Ddit4l</i> differ significantly (<i>Ccl21</i> 0.372, p=0.01; <i>Ddit4l</i> 0.275, p=0.04). <i>Ddit4l</i> encodes a protein involved in DNA-damage and hypoxia-induced cell death whilst disruption of the CCL21 pathway in mice improved myocardial dysfunction suggesting that CCL21 levels actively impact upon cardiac pathology. These results support the hypothesis that <i>Ccl21</i> and <i>Ddit4l</i> are positively regulated by <i>Pitx2c</i>, and may suggest that <i>Pitx2c</i> maintains leftness in the adult atrium; hence the contribution of these genes to AF pathology warrants further investigation.

Candidate Placental Biomarkers for Intrauterine Alcohol Exposure

Fetal alcohol spectrum disorder (FASD) is a leading cause of nongenetic mental retardation and other neurodevelopmental deficits. Earlier diagnosis of FASD would greatly improve prognosis for individuals and families affected by this disorder. Here, we identify candidate placental biomarkers in an animal model of FASD that recapitulates many aspects of human FASD. Pregnant Sprague-Dawley (SD) females were assigned to 1 of 3 diet groups on gestation day 8 (G8): Ethanol (E), Pair-fed (PF) or Control (C). E dams received ethanol-containing liquid diet and PF dams received isocaloric liquid diet in an amount that matched the paired E dam's diet consumption the previous day. Control dams received laboratory chow and water ad libitum. Whole placentae from individual fetuses were collected on gestational day 21 (G21) for analyses. Western blotting and quantitative real-time RT-PCR were used to measure protein and mRNA levels of placental iodothyronine deiodinase III (Dio3), thyroid hormone receptor α1 (TRα1), and glucocorticoid receptor (GR). Placental mRNA levels of insulin-like growth factor 2 (Igf-2), pleckstrin homology-like domain family A member 2 (Phlda2), and cyclin-dependent kinase inhibitor 1C (Cdkn1c) were also measured. Placental protein and mRNA levels from ethanol (E)-consuming dams showed the following changes: increased Dio3, decreased TRα1, and decreased GR compared to both C and PF dams. Placental mRNA levels of intrauterine growth restriction (IUGR) markers Igf-2, Phlda2, and Cdkn1c were altered similarly in PF and E dams. We propose the specific pattern of increased Dio3 and decreased TRα1 and GR protein levels in the placenta as selective biomarker for intrauterine alcohol exposure.

A Novel Pleckstrin Homology Domain-containing Protein Enhances Insulin-stimulated Akt Phosphorylation and GLUT4 Translocation in Adipocytes

Protein kinase B/Akt protein kinases control an array of diverse functions, including cell growth, survival, proliferation, and metabolism. We report here the identification of pleckstrin homology-like domain family B member 1 (PHLDB1) as an insulin-responsive protein that enhances Akt activation. PHLDB1 contains a pleckstrin homology domain, which we show binds phosphatidylinositol PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3, as well as a Forkhead-associated domain and coiled coil regions. PHLDB1 expression is increased during adipocyte differentiation, and it is abundant in many mouse tissues. Both endogenous and HA- or GFP-tagged PHLDB1 displayed a cytoplasmic disposition in unstimulated cultured adipocytes but translocated to the plasma membrane in response to insulin. Depletion of PHLDB1 by siRNA inhibited insulin stimulation of Akt phosphorylation but not tyrosine phosphorylation of IRS-1. RNAi-based silencing of PHLDB1 in cultured adipocytes also attenuated insulin-stimulated deoxyglucose transport and Myc-GLUT4-EGFP translocation to the plasma membrane, whereas knockdown of the PHLDB1 isoform PHLDB2 failed to attenuate insulin-stimulated deoxyglucose transport. Furthermore, adenovirus-mediated expression of PHLDB1 in adipocytes enhanced insulin-stimulated Akt and p70 S6 kinase phosphorylation, as well as GLUT4 translocation. These results indicate that PHLDB1 is a novel modulator of Akt protein kinase activation by insulin.

Maternal protein restriction with or without folic acid supplementation during pregnancy alters the hepatic transcriptome in adult male rats

Feeding pregnant rats a protein-restricted (PR) diet induces altered expression of candidate genes in the liver of the adult offspring, which can be prevented by supplementation of the PR diet with folic acid (PRF). We investigated the effect of maternal nutrition during pregnancy on the liver transcriptome in their adult male offspring. Pregnant rats were fed control, PR or PRF diets. Male offspring were killed on day 84. The liver transcriptome was analysed by microarray (six livers per maternal dietary group) followed by post hoc analysis of relative mRNA levels and gene ontology. These results were confirmed for selected genes by real-time RT-PCR. There were 311 genes that differed significantly ( >or= 1.5-fold change; P < 0.05) between PR offspring (222 increased) and control offspring, while 191 genes differed significantly between PRF offspring (forty-five increased) compared with offspring of control dams. There were sixteen genes that were significantly altered in both PR and PRF offspring compared with controls. Ion transport, developmental process, and response to reactive oxygen species (RROS) and steroid hormone response (SHR) ontologies were altered in PR offspring. Folic acid supplementation prevented changes within RROS and SHR response pathways, but not in ion transport or developmental process. There was no effect of maternal PR on mRNA expression of imprinted genes. Insulin 1 and Pleckstrin homology-like domain family A member 2 were increased significantly in PRF compared with PR offspring. The present findings show that the pattern of induced changes in the adult liver transcriptome were dependent on maternal protein and folic acid intakes during pregnancy.