A transformed view of cyclosporine

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62591/1/397471a0.pd

Mutations in the 3'-untranslated region of GATA4 as molecular hotspots for congenital heart disease (CHD)

<p>Abstract</p> <p>Background</p> <p>The 3'-untranslated region (3'-UTR) of mRNA contains regulatory elements that are essential for the appropriate expression of many genes. These regulatory elements are involved in the control of nuclear transport, polyadenylation status, subcellular targetting as well as rates of translation and degradation of mRNA. Indeed, 3'-UTR mutations have been associated with disease, but frequently this region is not analyzed. To gain insights into congenital heart disease (CHD), we have been analyzing cardiac-specific transcription factor genes, including <it>GATA4</it>, which encodes a zinc finger transcription factor. Germline mutations in the coding region of <it>GATA4 </it>have been associated with septation defects of the human heart, but mutations are rather rare. Previously, we identified 19 somatically-derived zinc finger mutations in diseased tissues of malformed hearts. We now continued our search in the 609 bp 3'-UTR region of <it>GATA4 </it>to explore further molecular avenues leading to CHD.</p> <p>Methods</p> <p>By direct sequencing, we analyzed the 3'-UTR of <it>GATA4 </it>in DNA isolated from 68 formalin-fixed explanted hearts with complex cardiac malformations encompassing ventricular, atrial, and atrioventricular septal defects. We also analyzed blood samples of 12 patients with CHD and 100 unrelated healthy individuals.</p> <p>Results</p> <p>We identified germline and somatic mutations in the 3'-UTR of <it>GATA4</it>. In the malformed hearts, we found nine frequently occurring sequence alterations and six dbSNPs in the 3'-UTR region of <it>GATA4</it>. Seven of these mutations are predicted to affect RNA folding. We also found further five nonsynonymous mutations in exons 6 and 7 of <it>GATA4</it>. Except for the dbSNPs, analysis of tissue distal to the septation defect failed to detect sequence variations in the same donor, thus suggesting somatic origin and mosaicism of mutations. In a family, we observed c.+119A > T in the 3'-UTR associated with ASD type II.</p> <p>Conclusion</p> <p>Our results suggest that somatic <it>GATA4 </it>mutations in the 3'-UTR may provide an additional molecular rationale for CHD.</p

Identification and Characterization of a Mef2 Transcriptional Activator in Schistosome Parasites

Myocyte enhancer factor 2 protein (Mef2) is an evolutionarily conserved activator of transcription that is critical to induce and control complex processes in myogenesis and neurogenesis in vertebrates and insects, and osteogenesis in vertebrates. In Drosophila, Mef2 null mutants are unable to produce differentiated muscle cells, and in vertebrates, Mef2 mutants are embryonic lethal. Schistosome worms are responsible for over 200 million cases of schistosomiasis globally, but little is known about early development of schistosome parasites after infecting a vertebrate host. Understanding basic schistosome development could be crucial to delineating potential drug targets. Here, we identify and characterize Mef2 from the schistosome worm Schistosoma mansoni (SmMef2). We initially identified SmMef2 as a homolog to the yeast Mef2 homolog, Resistance to Lethality of MKK1P386 overexpression (Rlm1), and we show that SmMef2 is homologous to conserved Mef2 family proteins. Using a genetics approach, we demonstrate that SmMef2 is a transactivator that can induce transcription of four separate heterologous reporter genes by yeast one-hybrid analysis. We also show that Mef2 is expressed during several stages of schistosome development by quantitative PCR and that it can bind to conserved Mef2 DNA consensus binding sequences

Congenital Heart Disease–Causing Gata4 Mutation Displays Functional Deficits In Vivo

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans

Genome-wide association study identifies single-nucleotide polymorphism in KCNB1 associated with left ventricular mass in humans: The HyperGEN Study

<p>Abstract</p> <p>Background</p> <p>We conducted a genome-wide association study (GWAS) and validation study for left ventricular (LV) mass in the Family Blood Pressure Program – HyperGEN population. LV mass is a sensitive predictor of cardiovascular mortality and morbidity in all genders, races, and ages. Polymorphisms of candidate genes in diverse pathways have been associated with LV mass. However, subsequent studies have often failed to replicate these associations. Genome-wide association studies have unprecedented power to identify potential genes with modest effects on left LV mass. We describe here a GWAS for LV mass in Caucasians using the Affymetrix GeneChip Human Mapping 100 k Set. Cases (N = 101) and controls (N = 101) were selected from extreme tails of the LV mass index distribution from 906 individuals in the HyperGEN study. Eleven of 12 promising (<it>Q </it>< 0.8) single-nucleotide polymorphisms (SNPs) from the genome-wide study were successfully genotyped using quantitative real time PCR in a validation study.</p> <p>Results</p> <p>Despite the relatively small sample, we identified 12 promising SNPs in the GWAS. Eleven SNPs were successfully genotyped in the validation study of 704 Caucasians and 1467 African Americans; 5 SNPs on chromosomes 5, 12, and 20 were significantly (<it>P </it>≤ 0.05) associated with LV mass after correction for multiple testing. One SNP (rs756529) is intragenic within <it>KCNB1</it>, which is dephosphorylated by calcineurin, a previously reported candidate gene for LV hypertrophy within this population.</p> <p>Conclusion</p> <p>These findings suggest <it>KCNB1 </it>may be involved in the development of LV hypertrophy in humans.</p

Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice

<p>Abstract</p> <p>Background</p> <p>The transcription factor NFAT5/TonEBP regulates the response of mammalian cells to hypertonicity. However, little is known about the physiopathologic tonicity thresholds that trigger its transcriptional activity in primary cells. Wilkins et al. recently developed a transgenic mouse carrying a luciferase reporter (9xNFAT-Luc) driven by a cluster of NFAT sites, that was activated by calcineurin-dependent NFATc proteins. Since the NFAT site of this reporter was very similar to an optimal NFAT5 site, we tested whether this reporter could detect the activation of NFAT5 in transgenic cells.</p> <p>Results</p> <p>The 9xNFAT-Luc reporter was activated by hypertonicity in an NFAT5-dependent manner in different types of non-transformed transgenic cells: lymphocytes, macrophages and fibroblasts. Activation of this reporter by the phorbol ester PMA plus ionomycin was independent of NFAT5 and mediated by NFATc proteins. Transcriptional activation of NFAT5 in T lymphocytes was detected at hypertonic conditions of 360–380 mOsm/kg (isotonic conditions being 300 mOsm/kg) and strongly induced at 400 mOsm/kg. Such levels have been recorded in plasma in patients with osmoregulatory disorders and in mice deficient in aquaporins and vasopressin receptor. The hypertonicity threshold required to activate NFAT5 was higher in bone marrow-derived macrophages (430 mOsm/kg) and embryonic fibroblasts (480 mOsm/kg). Activation of the 9xNFAT-Luc reporter by hypertonicity in lymphocytes was insensitive to the ERK inhibitor PD98059, partially inhibited by the PI3-kinase inhibitor wortmannin (0.5 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages.</p> <p>Conclusion</p> <p>Our results indicate that NFAT5 is a sensitive responder to pathologic increases in extracellular tonicity in T lymphocytes. Activation of NFAT5 by hypertonicity in lymphocytes was mediated by a combination of signaling pathways that differed from those required in other cell types. We propose that the 9xNFAT-Luc transgenic mouse model might be useful to study the physiopathological regulation of both NFAT5 and NFATc factors in primary cells.</p

Simultaneous expression of Oct4 and genes of three germ layers in single cell-derived multipotent adult progenitor cells

Future application of adult stem cells in clinical therapies largely depends on the successful isolation of homogeneous stem cells with high plasticity. Multipotent adult progenitor cells (MAPCs) are thought to be a more primitive stem cell population capable of extensive in vitro proliferation with no senescence or loss of differentiation capability. The present study was aimed to find a less complicated and more economical protocol for obtaining single cell-derived MAPCs and understand the molecule mechanism of multi-lineage differentiation of MAPCs. We successfully obtained a comparatively homogeneous population of MAPCs and confirmed that single cell-derived MAPCs were able to transcribe Oct4 and genes of three germ layers simultaneously, and differentiate into multiple lineages. Our observations suggest that single cell-derived MAPCs under appropriate circumstances could maintain not only characteristics of stem cells but multi-lineage differentiation potential through quantitative modulation of corresponding regulating gene expression, rather than switching on expression of specific genes

BNP controls early load-dependent regulation of SERCA through calcineurin

Heart failure is characterised by reduced expression of sarcoplasmic reticulum calcium-ATPase (SERCA) and increased expression of B-type natriuretic peptide (BNP). The present study was performed to investigate causality of this inverse relationship under in vivo conditions in the transversal aortic constriction mouse model (TAC). Left ventricular SERCA-mRNA expression was significantly upregulated in TAC by 32% after 6 h, but not different from sham after 24 h. Serum proANP and BNP levels were increased in TAC after 24 h (BNP +274%, p < 0.01; proANP +60%, p < 0.05), but only proANP levels were increased after 6 h (+182%, p < 0.01). cGMP levels were only increased 24 h after TAC (+307%, p < 0.01), but not 6 h after TAC. BNP infusion inhibited the increase in SERCA expression 6 h after TAC. In BNP-receptor-knockout animals (GC-A), the expression of SERCA was still significantly increased 24 h after TAC at the mRNA level by 35% (p < 0.05), as well as at the protein level by 25% (p < 0.05). MCIP expression as an indicator of calcineurin activity was regulated in parallel to SERCA after 6 and 24 h. MCIP-mRNA was increased by 333% 6 h after TAC, but not significantly different from sham after 24 h. In the GC-A-KO mice, MCIP-mRNA was significantly increased in TAC compared to WT after 24 h. In mice with BNP infusion, MCIP was significantly lower 6 h after TAC compared to control animals. In conclusion, mechanical load leads to an upregulation of SERCA expression. This is followed by upregulation of natriuretic peptides with subsequent suppression of SERCA upregulation. Elevated natriuretic peptides may suppress SERCA expression by inhibition of calcineurin activity via activation of GC-A

Syndecan-4 Is Essential for Development of Concentric Myocardial Hypertrophy via Stretch-Induced Activation of the Calcineurin-NFAT Pathway

Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a co-receptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4−/− mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurin-nuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4−/−-NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pull-down experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased syndecan-4 levels with decreased pS179 which was associated with increased NFAT activation. In conclusion, our data show that syndecan-4 is essential for compensatory hypertrophy in the pressure overloaded heart. Specifically, syndecan-4 regulates stretch-induced activation of the calcineurin-NFAT pathway in cardiomyocytes. Thus, our data suggest that manipulation of syndecan-4 may provide an option for therapeutic modulation of calcineurin-NFAT signaling

TrpC3 Regulates Hypertrophy-Associated Gene Expression without Affecting Myocyte Beating or Cell Size

Pathological cardiac hypertrophy is associated with an increased risk of heart failure and cardiovascular mortality. Calcium (Ca2+) -regulated gene expression is essential for the induction of hypertrophy, but it is not known how myocytes distinguish between the Ca2+ signals that regulate contraction and those that lead to cardiac hypertrophy. We used in vitro neonatal rat ventricular myocytes to perform an RNA interference (RNAi) screen for ion channels that mediate Ca2+-dependent gene expression in response to hypertrophic stimuli. We identified several ion channels that are linked to hypertrophic gene expression, including transient receptor potential C3 (TrpC3). RNAi-mediated knockdown of TrpC3 decreases expression of hypertrophy-associated genes such as the A- and B-type natriuretic peptides (ANP and BNP) in response to numerous hypertrophic stimuli, while TrpC3 overexpression increases BNP expression. Furthermore, stimuli that induce hypertrophy dramatically increase TrpC3 mRNA levels. Importantly, whereas TrpC3-knockdown strongly reduces gene expression associated with hypertrophy, it has a negligible effect on cell size and on myocyte beating. These results suggest that Ca2+ influx through TrpC3 channels increases transcription of genes associated with hypertrophy but does not regulate the signaling pathways that control cell size or contraction. Thus TrpC3 may represent an important therapeutic target for the treatment of cardiac hypertrophy and heart failure