CUGBP1 and HuR regulate E-cadherin translation by altering recruitment of E-cadherin mRNA to processing bodies and modulate epithelial barrier function.

The effectiveness and stability of epithelial barrier depend on apical junctional complexes, which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs, and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the posttranscriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HU antigen R (HuR) are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs and therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.

[Effect of external retinoic acid on Tbx1 gene during zebrafish embryogenesis].

OBJECTIVE: DiGeorge/del22q11 syndrome is one of the most common genetic causes of outflow tract and aortic arch defects in human. DiGeorge/del22q11 is thought to involve an embryonic defect restricted to the pharyngeal arches and the corresponding pharyngeal pouches. Previous studies have evidenced that retinoic acid (RA) signaling is definitely indispensable for the development of the pharyngeal arches. Tbx1, one of the T-box containing genes, is proved to be the most attractive candidate gene for DiGeorge/del22q11 syndrome. However, the interaction between RA and Tbx1 has not been fully investigated. Exploring the interaction will contribute to discover the molecular pathways disrupted in DiGeorge/del22q11 syndrome, and will also be essential for understanding genetic basis for congenital heart disease. It now seems possible that genes and molecular pathways disrupted in DiGeorge syndrome will also account for some isolated cases of congenital heart disease. Accordingly, the present study aimed to extensively study the effects of external RA on the cardiac development and Tbx1 expression during zebrafish embryogenesis. METHODS: The chemical genetics approach was applied by treating zebrafish embryos with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA at 12.5 hour post fertilization (hpf). The expression patterns of Tbx1 were monitored by whole-mount in situ hybridization and quantitative real-time RT-PCR, respectively. RESULTS: The zebrafish embryos treated with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA for 1.5 h at 12.5 hpf exhibited selective defects of abnormal heart tube. The results of whole-mount in situ hybridization with Tbx1 RNA probe showed that Tbx1 was expressed in cardiac region, pharyngeal arches and otic vesicle during zebrafish embryogenesis. RA treatment led to a distinct spatio-temporal expression pattern for Tbx1 from that in wild type embryo. The real-time PCR analysis showed that Tbx1 expression levels were markedly reduced by RA treatment. Tbx1 expression in the pharyngeal arches and heart were obviously down regulated compared to the wild type embryos. In contrast to 5 x 10(-8) mol/L RA-treated groups, 10(-7) mol/L RA caused a more severe effect on the Tbx1 expression level. CONCLUSION: These results suggested that there was a genetic link between RA and Tbx1 during development of zebrafish embryo. RA could produce an altered Tbx1 expression pattern in zebrafish. RA may regulate the Tbx1 expression in a dose-dependant manner. RA could represent a major epigenetic factor to cause abnormal expression of Tbx1, secondarily, disrupt the pharyngeal arch and heart development.

Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development.

BACKGROUND: Folic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of folic acid performing its biological function. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed the abnormal cardiac phenotypes in dihydrofolate reductase (DHFR) gene knock-down zebrafish embryos, investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2 (HAND2) and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations. METHODS: Morpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression. RESULTS: DHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 was obviously reduced in DHFR knock-down embryos (P < 0.05). Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-down embryos. CONCLUSIONS: DHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.

Bone morphogenetic protein-2 acts upstream of myocyte-specific enhancer factor 2a to control embryonic cardiac contractility.

OBJECTIVE: Cardiac contractility is regulated tightly as an extrinsic and intrinsic homeostatic mechanism to the heart. The molecular basis of the intrinsic system is largely unknown. Here, we test the hypothesis that bone morphogenetic protein-2 (BMP-2) mediates embryonic cardiac contractility upstream of myocyte-specific enhancer factor 2A (MEF2A). METHODS: The BMP-2 and MEF2A expression pattern was analyzed by RT-PCR, Western blotting, whole-mount in situ hybridization, and an in vivo transgenic approach. The cardiac phenotype of BMP-2 and MEF2A knock-down zebrafish embryos was analysed. Cardiac contractions were recorded with a video camera. Myofibrillar organization was observed with transmission electron microscopy. Gene expression profiles were performed by quantitative real-time PCR analysis. RESULTS: We demonstrate that BMP-2 and MEF2A are co-expressed in embryonic and neonatal cardiac myocytes. Furthermore, we provide evidence that BMP-2 is required for cardiac contractility in vitro and in vivo and that MEF2A expression can be activated by BMP-2 signaling in neonatal cardiomyocytes. BMP-2 is involved in the assembly of the cardiac contractile apparatus. Finally, we find that exogenous MEF2A is sufficient to rescue ventricular contractility defects in the absence of BMP-2 function. CONCLUSIONS: In all, these observations indicate that BMP-2 and MEF2A are key components of a pathway that controls the cardiac ventricular contractility and suggest that the BMP2-MEF2A pathway can offer new opportunities for the treatment of heart failure.

Requirements of myocyte-specific enhancer factor 2A in zebrafish cardiac contractility.

Myocyte-specific enhancer factor 2A (MEF2A) regulates a broad range of fundamental cellular processes including cell division, differentiation and death. Here, we tested the hypothesis that MEF2A is required in cardiac contractility employing zebrafish as a model organism. MEF2A is highly expressed in heart as well as somites during zebrafish embryogenesis. Knock-down of MEF2A in zebrafish impaires the cardiac contractility and results in sarcomere assembly defects. Dysregulation of cardiac genes in MEF2A morphants suggests that sarcomere assembly disturbances account for the cardiac contractile deficiency. Our studies suggested that MEF2A is essential in cardiac contractility.