Copy-Number Variation of the Glucose Transporter Gene SLC2A3 and Congenital Heart Defects in the 22q11.2 Deletion Syndrome

The 22q11.2 deletion syndrome (22q11DS; velocardiofacial/DiGeorge syndrome; VCFS/DGS) is the most common microdeletion syndrome and the phenotypic presentation is highly variable. Approximately 65% of individuals with 22q11DS have a congenital heart defect (CHD), mostly of the conotruncal type, and/or an aortic arch defect. The etiology of this phenotypic variability is not currently known. We hypothesized that copy-number variants (CNVs) outside the 22q11.2 deleted region might increase the risk of being born with a CHD in this sensitized population. Genotyping with Affymetrix SNP Array 6.0 was performed on two groups of subjects with 22q11DS separated by time of ascertainment and processing. CNV analysis was completed on a total of 949 subjects (cohort 1, n = 562; cohort 2, n = 387), 603 with CHDs (cohort 1, n = 363; cohort 2, n = 240) and 346 with normal cardiac anatomy (cohort 1, n = 199; cohort 2, n = 147). Our analysis revealed that a duplication of SLC2A3 was the most frequent CNV identified in the first cohort. It was present in 18 subjects with CHDs and 1 subject without (p = 3.12 × 10⁻³, two-tailed Fisher’s exact test). In the second cohort, the SLC2A3 duplication was also significantly enriched in subjects with CHDs (p = 3.30 × 10⁻², two-tailed Fisher’s exact test). The SLC2A3 duplication was the most frequent CNV detected and the only significant finding in our combined analysis (p = 2.68 × 10⁻⁴, two-tailed Fisher’s exact test), indicating that the SLC2A3 duplication might serve as a genetic modifier of CHDs and/or aortic arch anomalies in individuals with 22q11DS.     

Transgenic Expression of MicroRNA-185 Causes a Developmental Arrest of T Cells by Targeting Multiple Genes Including Mzb1

miR-185 is a microRNA (miR) that targets Bruton''s tyrosine kinase in B cells, with reductions in miR-185 linked to B cell autoantibody production. In hippocampal neurons, miR-185 targets both sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and a novel Golgi inhibitor. This miR is haploinsufficient in 90–95% of individuals with chromosome 22q11.2 deletion syndrome, patients who can present with immune, cardiac, and parathyroid problems, learning disorders, and a high incidence of schizophrenia in adults. The reduced levels of miR-185 in neurons cause presynaptic neurotransmitter release. Many of the 22q11.2 deletion syndrome patients have a thymic hypoplasia, which results in a peripheral T cell lymphopenia and unusual T helper cell skewing. The molecular targets of miR-185 in thymocytes are unknown. Using an miR-185 T cell transgenic approach, increasing levels of miR-185 attenuated T cell development at the T cell receptor β (TCRβ) selection checkpoint and during positive selection. This caused a peripheral T cell lymphopenia. Mzb1, Nfatc3, and Camk4 were identified as novel miR-185 targets. Elevations in miR-185 enhanced TCR-dependent intracellular calcium levels, whereas a knockdown of miR-185 diminished these calcium responses. These effects concur with reductions in Mzb1, an endoplasmic reticulum calcium regulator. Consistent with their haploinsufficiency of miR-185, Mzb1 levels were elevated in thymocyte extracts from several 22q11.2 deletion syndrome patients. Our findings indicate that miR-185 regulates T cell development through its targeting of several mRNAs including Mzb1.     

The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain

In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C₅ acyl side chain of the C₂₄ steroid compound cholate involves the C₂₂ intermediate 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C₂₄ steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD⁺ as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.     

Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) Signaling Regulates Mitochondrial Biogenesis and Respiration via Estrogen-related Receptor α (ERRα)

Mitochondrial abnormalities are associated with cancer development, yet how oncogenic signals affect mitochondrial functions has not been fully understood. In this study, we investigate the relationship between mitochondrial alterations and PI3K/protein kinase B (AKT) signaling activation using hepatocytes and liver tissues as our experimental models. We show here that liver-specific deletion of Pten, which leads to activation of PI3K/AKT, is associated with elevated oxidative stress, increased mitochondrial mass, and augmented respiration accompanied by enhanced glycolysis. Consistent with these observations, estrogen-related receptor α (ERRα), an orphan nuclear receptor known for its role in mitochondrial biogenesis, is up-regulated in the absence of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Our pharmacological and genetic studies show that PI3K/AKT activity regulates the expression of ERRα and mitochondrial biogenesis/respiration. Furthermore, cAMP-response element-binding protein, as a downstream target of AKT, plays a role in the regulation of ERRα, independent of PKA signaling. ERRα regulates reactive oxygen species production, and ERRα knockdown attenuates proliferation and colony-forming potential in Pten-null hepatocytes. Finally, analysis of clinical datasets from liver tissues showed a negative correlation between expressions of ERRα and PTEN in patients with liver cancer. Therefore, this study has established a previously unrecognized link between a growth signal and mitochondrial metabolism.     

Common Polymorphisms Influencing Serum Uric Acid Levels Contribute to Susceptibility to Gout,but Not to Coronary Artery Disease

Background

Recently, a large meta-analysis including over 28,000 participants identified nine different loci with association to serum uric acid (UA) levels. Since elevated serum UA levels potentially cause gout and are a possible risk factor for coronary artery disease (CAD) and myocardial infarction (MI), we performed two large case-control association analyses with participants from the German MI Family Study. In the first study, we assessed the association of the qualitative trait gout and ten single nucleotide polymorphisms (SNP) markers that showed association to UA serum levels. In the second study, the same genetic polymorphisms were analyzed for association with CAD.

Methods and Findings

A total of 683 patients suffering from gout and 1,563 healthy controls from the German MI Family Study were genotyped. Nine SNPs were identified from a recently performed genome-wide meta-analysis on serum UA levels (rs12129861, rs780094, rs734553, rs2231142, rs742132, rs1183201, rs12356193, rs17300741 and rs505802). Additionally, the marker rs6855911 was included which has been associated with gout in our cohort in a previous study. SNPs rs734553 and rs6855911, located in SLC2A9, and SNP rs2231142, known to be a missense polymorphism in ABCG2, were associated with gout (p = 5.6*10⁻⁷, p = 1.1*10⁻⁷, and p = 1.3*10⁻³, respectively). Other SNPs in the genes PDZK1, GCKR, LRRC16A, SLC17A1-SLC17A3, SLC16A9, SLC22A11 and SLC22A12 failed the significance level. None of the ten markers were associated with risk to CAD in our study sample of 1,473 CAD cases and 1,241 CAD-free controls.

Conclusion

SNP markers in SLC2A9 and ABCG2 genes were found to be strongly associated with the phenotype gout. However, not all SNP markers influencing serum UA levels were also directly associated with the clinical manifestation of gout in our study sample. In addition, none of these SNPs showed association with the risk to CAD in the German MI Family Study.     

PGC-1α limits angiotensin II-induced rat vascular smooth muscle cells proliferation via attenuating NOX1-mediated generation of reactive oxygen species

AngII (angiotensin II)-induced excessive ROS (reactive oxygen species) generation and proliferation of VSMCs (vascular smooth muscle cells) is a critical contributor to the pathogenesis of atherosclerosis. PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-1α] is involved in the regulation of ROS generation, VSMC proliferation and energy metabolism. The aim of the present study was to investigate whether PGC-1α mediates AngII-induced ROS generation and VSMC hyperplasia. Our results showed that the protein content of PGC-1α was negatively correlated with an increase in cell proliferation and migration induced by AngII. Overexpression of PGC-1α inhibited AngII-induced proliferation and migration, ROS generation and NADPH oxidase activity in VSMCs. Conversely, Ad-shPGC-1α (adenovirus-mediated PGC-1α-specific shRNA) led to the opposite effects. Furthermore, the stimulatory effect of Ad-shPGC-1α on VSMC proliferation was significantly attenuated by antioxidant and NADPH oxidase inhibitors. Analysis of several key subunits of NADPH oxidase (Rac1, p22^phox, p40^phox, p47^phox and p67^phox) and mitochondrial ROS revealed that these mechanisms were not responsible for the observed effects of PGC-1α. However, we found that overexpression of PGC-1α promoted NOX1 degradation through the proteasome degradation pathway under AngII stimulation and consequently attenuated NOX1 (NADPH oxidase 1) expression. These alterations underlie the inhibitory effect of PGC-1α on NADPH oxidase activity. Our data support a critical role for PGC-1α in the regulation of proliferation and migration of VSMCs, and provide a useful strategy to protect vessels against atherosclerosis.     

A Solute Carrier Family 22 Member 3 Variant rs3088442 G→A Associated with Coronary Heart Disease Inhibits Lipopolysaccharide-induced Inflammatory Response

Recent genome-wide association studies have identified single-nucleotide polymorphism (SNPs) within the SLC22A3 (solute carrier family 22 member 3) gene associated with coronary heart disease (CHD) in the Caucasian population. We performed molecular analysis to investigate the potential role of SLC22A3 variants in CHD. Our study showed that the common polymorphism rs3088442 G→A, which is localized in the 3′ UTR of the SLC22A3 gene, was associated with a decreased risk of CHD in the Chinese population by a case control study. In silico analysis indicated that G→A substitution of SNP rs3088442 created a putative binding site for miR-147 in the SLC22A3 mRNA. By overexpressing miR-147 or inhibiting endogenous miR-147, we demonstrated that SNP rs3088442 G→A recruited miR-147 to inhibit SLC22A3 expression. Moreover, SLC22A3 deficiency significantly decreased LPS-induced monocytic inflammatory response by interrupting NF-κB and MAPK signaling cascades in a histamine-dependent manner. Notably, the expression of SLC22A3^A was also suppressed by LPS stimulus. Our findings might indicate a negative feedback mechanism against inflammatory response by which SLC22A3 polymorphisms decreased the risk of CHD.     

Estrogen Receptor (ER)α-regulated Lipocalin 2 Expression in Adipose Tissue Links Obesity with Breast Cancer Progression

Obesity is associated with increased breast cancer (BrCA) incidence. Considering that inactivation of estrogen receptor (ER)α promotes obesity and metabolic dysfunction in women and female mice, understanding the mechanisms and tissue-specific sites of ERα action to combat metabolic-related disease, including BrCA, is of clinical importance. To study the role of ERα in adipose tissue we generated fat-specific ERα knock-out (FERKO) mice. Herein we show that ERα deletion increased adipocyte size, fat pad weight, and tissue expression and circulating levels of the secreted glycoprotein, lipocalin 2 (Lcn2), an adipokine previously associated with BrCA development. Chromatin immunoprecipitation and luciferase reporter studies showed that ERα binds the Lcn2 promoter to repress its expression. Because adipocytes constitute an important cell type of the breast microenvironment, we examined the impact of adipocyte ERα deletion on cancer cell behavior. Conditioned medium from ERα-null adipocytes and medium containing pure Lcn2 increased proliferation and migration of a subset of BrCA cells in culture. The proliferative and promigratory effects of ERα-deficient adipocyte-conditioned medium on BrCA cells was reversed by Lcn2 deletion. BrCA cell responsiveness to exogenous Lcn2 was heightened in cell types where endogenous Lcn2 expression was minimal, but components of the Lcn2 signaling pathway were enriched, i.e. SLC22A17 and 3-hydroxybutyrate dehydrogenase (BDH2). In breast tumor biopsies from women diagnosed with BrCA we found that BDH2 expression was positively associated with adiposity and circulating Lcn2 levels. Collectively these data suggest that reduction of ERα expression in adipose tissue promotes adiposity and is linked with the progression and severity of BrCA via increased adipocyte-specific Lcn2 production and enhanced tumor cell Lcn2 sensitivity.     

Heat Shock Protein 22 (Hsp22) Regulates Oxidative Phosphorylation upon Its Mitochondrial Translocation with the Inducible Nitric Oxide Synthase in Mammalian Heart

Objectives

Stress-inducible heat shock protein 22 (Hsp22) confers protection against ischemia through induction of the inducible isoform of nitric oxide synthase (iNOS). Hsp22 overexpression in vivo stimulates cardiac mitochondrial respiration, whereas Hsp22 deletion in vivo significantly reduces respiration. We hypothesized that Hsp22-mediated regulation of mitochondrial function is dependent upon its mitochondrial translocation together with iNOS.

Methods and Results

Adenoviruses harboring either the full coding sequence of Hsp22 (Ad-WT-Hsp22) or a mutant lacking a N-terminal 20 amino acid putative mitochondrial localization sequence (Ad-N20-Hsp22) were generated, and infected in rat neonatal cardiomyocytes. Compared to β-Gal control, WT-Hsp22 accumulated in mitochondria by 2.5 fold (P<0.05) and increased oxygen consumption rates by 2-fold (P<0.01). This latter effect was abolished upon addition of the selective iNOS inhibitor, 1400W. Ad-WT-Hsp22 significantly increased global iNOS expression by about 2.5-fold (P<0.01), and also increased iNOS mitochondrial localization by 4.5 fold vs β-gal (P<0.05). Upon comparable overexpression, the N20-Hsp22 mutant did not show significant mitochondrial translocation or stimulation of mitochondrial respiration. Moreover, although N20-Hsp22 did increase global iNOS expression by 4.6-fold, it did not promote iNOS mitochondrial translocation.

Conclusion

Translocation of both Hsp22 and iNOS to the mitochondria is necessary for Hsp22-mediated stimulation of oxidative phosphorylation.     

The m6A methyltransferase METTL3 modifies PGC-1α mRNA promoting mitochondrial dysfunction and oxLDL-induced inflammation in monocytes

Mitochondrial biogenesis and energy metabolism are essential for regulating the inflammatory state of monocytes. This state is partially controlled by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a coactivator that regulates mitochondrial biogenesis and energy metabolism. Disruption of these processes can also contribute to the initiation of chronic inflammatory diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis. Methyltransferase-like 3 (METTL3)-dependent N⁶-methyladenosine (m⁶A) methylation has recently been shown to regulate a variety of inflammatory processes. However, the role of m⁶A mRNA methylation in affecting mitochondrial metabolism in monocytes under inflammation is unclear, nor is there an established relationship between m⁶A methylation and PGC-1α. In this study, we identified a novel mechanism by which METTL3 acts during oxidized low-density lipoprotein (oxLDL)-induced monocyte inflammation, where METTL3 and YTH N⁶-methyladenosine RNA binding protein 2 (YTHDF2) cooperatively modify PGC-1α mRNA, mediating its degradation, decreasing PGC-1α protein levels, and thereby enhancing the inflammatory response. METTL3 coordinated with YTHDF2 to suppress the expression of PGC-1α, as well as that of cytochrome c (CYCS) and NADH:ubiquinone oxidoreductase subunit C2 (NDUFC2) and reduced ATP production and oxygen consumption rate (OCR). This subsequently increased the accumulation of cellular and mitochondrial reactive oxygen species (ROS) and the levels of proinflammatory cytokines in inflammatory monocytes. These data may provide new insights into the role of METTL3-dependent m⁶A modification of PGC-1α mRNA in the monocyte inflammation response. These data also contribute to a more comprehensive understanding of the pathogenesis of monocyte-macrophage inflammation-associated diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis.     

Nucleotide Sequence of the F(1)-ATPase alpha Subunit Gene from Maize Mitochondria

The α subunit of the F₁-ATPase complex of maize is a mitochondrial translational product, presumably encoded by the mitochondrial genome. Based on nucleotide and amino acid homology, we have identified a mitochondrial gene, designated atpα, that appears to code for the F₁-ATPase α subunit of Zea mays. The atpα gene is present as a single copy in the maize. Texas cytoplasm and is actively transcribed. The maize α polypeptide has a predicted length of 508 amino acids and a molecular mass of 55,187 daltons. Amino acid homologies between the maize mitochondrial α subunit and the tobacco chloroplast CF₁ and Escherichia coli α subunits are 54 and 51%, respectively. The origin of the atpα gene is discussed.