👤 Nobutaka Koibuchi

Thyroid hormones (THs) and estrogen (E2) play essential roles in neuronal differentiation and plasticity during brain development. S-equol, a plant-derived isoflavone metabolite, is a selective E2 receptor (ER) ligand that exhibits neurotrophic effects; however, its interaction with TH receptor (TR) signaling remains unclear. In this study, we investigated the effects of S-equol on TR Show less

The transcription factor CHF1/Hey2 has been implicated in a variety of cardiovascular developmental abnormalities including ventricular septal defect, deformed valves and cardiomyopathy. To date, its role in coronary vascular development remains unknown. We have found that KO mice developed coronary vascular abnormalities accompanied by a thin compact ventricular myocardium but grossly normal epicardial and subepicardial layers. The coronary vascular anomalies included dysmorphic large vessels and abnormal vascular structures at E15.5 and reduced recruitment of vascular smooth muscle cells into the coronary arteries at E18.5. In E18.5 KO hearts, the abnormal coronary veins demonstrated reduced expression of markers for vein identity. Whole-mount PECAM staining of the E18.5 KO hearts indicated that EphB4 negative vein networks were increased in the surface layers of the myocardium compared to those of the controls. CHF1/Hey2 was not expressed in the epicardium in vivo, and cultured epicardium-derived cells isolated from E12.5 wild-type mice showed no CHF1/Hey2 expression. KO mice with a myocardially expressed CHF1/Hey2 transgene partially rescued the vascular phenotypes. Quantitative RT-PCR analysis demonstrated that PDGF and Angiopoietin/Tie2 signaling pathways are altered in E12.5 KO hearts. Taken together, global CHF1/Hey2 deficiency caused impaired vascular formation, the reduced recruitment of vascular smooth muscle cells into coronary arteries and abnormally remodeled vein networks. These findings suggest that CHF1/Hey2 regulates the later steps of coronary vascular development in both a myocardial-dependent, non-cell autonomous fashion and likely a vascular cell-specific effect as well. Show less

We previously reported that mice lacking the hairy-related basic helix-loop-helix (bHLH) transcription factor CHF1/Hey2 develop a thin-walled left ventricle. To explore the basis for this phenotype, we examined regional gene expression patterns in the developing myocardium. We found that atrial natriuretic factor (ANF), which is normally expressed in the atria and trabeculae and is restricted from the developing compact myocardium beginning at embryonic day 13.5, is persistently expressed in the left ventricular compact myocardium of the knockout animals. We also examined the expression pattern of the T-box transcription factor Tbx5, a known regulator of ANF, and an additional Tbx5-dependent gene, connexin 40 (Cx40), both of which share a similar expression pattern to ANF during development. Tbx5 and Cx40 were similarly expressed ectopically in the compact myocardium of the CHF1/Hey2 knockout mouse. The atrial contractile genes mlc1a and mlc2a were also expressed ectopically in the left ventricular compact myocardium, providing evidence for a general dysregulation of atrial gene expression. Crossing of a myocardial-specific CHF1/Hey2 transgenic mouse with the knockouts led to rescue of the thin-walled myocardial phenotype and restoration of the normal patterns of gene expression. Myocardial cell proliferation, which has been shown previously to be suppressed by Tbx5, was also decreased in the knockout mice and rescued by the transgene. Our findings suggest that CHF1/Hey2 suppresses atrial identity in the left ventricular compact myocardium, facilitates myocardial proliferation by suppressing Tbx5, and thereby promotes proper ventricular myocardial maturation. Show less

The bHLH transcription factor CHF1/Hey2 has been previously shown to regulate neointimal formation after vascular injury, but the mechanisms have not been fully elucidated. The zinc-finger protein GATA-6 has also been shown to regulate vascular smooth-muscle phenotype through regulation of smooth-muscle contractile protein gene expression. To address the potential mechanisms by which CHF1/Hey2 regulates vascular smooth-muscle phenotype switching, we investigated the effect of CHF1/Hey2 on GATA-6-dependent smooth-muscle myosin heavy chain promoter activity. When cotransfected into NIH3T3 cells, CHF1/Hey2 reduced GATA-6-dependent activation of the promoter by 90%. Exogenous p300 was not sufficient to overcome this repression effect, demonstrating that the inhibitor effect did not involve coactivation by p300. Coimmunoprecipitation studies demonstrated that CHF1/Hey2 interacts directly with GATA-6. Mutational analysis demonstrated that the bHLH domain is required for transcriptional repression. Our findings highlight an important transcriptional mechanism by which CHF1/Hey2 may affect smooth-muscle cell phenotype. Show less

CHF1/Hey2 null mice generated in different laboratories have discrepant cardiovascular phenotypes. To determine the effect of genetic background on phenotype, we backcrossed our knockout strain more than eight generations to the inbred strains BALB/c and C57BL/6. Knockout mice on these backgrounds showed disparate phenotypes. Mice on both backgrounds demonstrated ventricular septal defects (VSDs), tricuspid stenosis and mitral valve thickening, but at varying frequencies, suggesting a general defect in endocardial cushion remodeling. Additional defects seen exclusively on the C57BL/6 background included biventricular wall thinning and left ventricular enlargement, implying a more severe myocardial defect than previously observed. In addition, aortas and pulmonary arteries from these null mice had thinner walls. Intercrossing of the CHF1/Hey2 null mice on a C57BL/6 background with a C57BL/6 MLC2v-CHF1/Hey2 transgenic line overexpressing CHF1/Hey2 in the atrial and ventricular myocardium also rescued the VSD and myocardial phenotypes, but did not affect vascular wall thickness. Our results indicate that CHF1/Hey2 provides an important myocardial signal to the endocardial cushion for proper septation and valve formation and also plays an important role in maturation of the myocardium and vasculature. Show less