Dax1 associates with Esrrb and regulates its function in embryonic stem cells.

Self-renewal capacity and pluripotency, which are controlled by the Oct3/4-centered transcriptional regulatory network, are major characteristics of embryonic stem (ES) cells. Nuclear hormone receptor Dax1 is one of the crucial factors in the network. Here, we identified an orphan nuclear receptor, Esrrb (estrogen-related receptor beta), as a Dax1-interacting protein. Interaction of Dax1 and Esrrb was mediated through LXXLL motifs of Dax1 and the activation- and ligand-binding domains of Esrrb. Furthermore, Esrrb enhanced the promoter activity of the Dax1 gene via direct binding to Esrrb-binding site 1 (ERRE1, where "ERRE" represents "Esrrb-responsive element") of the promoter. Expression of Dax1 was suppressed followed by Oct3/4 repression; however, overexpression of Esrrb maintained expression of Dax1 even in the absence of Oct3/4, indicating that Dax1 is a direct downstream target of Esrrb and that Esrrb can regulate Dax1 expression in an Oct3/4-independent manner. We also found that the transcriptional activity of Esrrb was repressed by Dax1. Furthermore, we revealed that Oct3/4, Dax1, and Esrrb have a competitive inhibition capacity for each complex. These data, together with previous findings, suggest that Dax1 functions as a negative regulator of Esrrb and Oct3/4, and these molecules form a regulatory loop for controlling the pluripotency and self-renewal capacity of ES cells.

Dax1 binds to Oct3/4 and inhibits its transcriptional activity in embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Transcription factor Oct3/4 is an indispensable factor in the self-renewal of ES cells. In this study, we searched for a protein that would interact with Oct3/4 in ES cells and identified an orphan nuclear hormone receptor, Dax1. The association of Dax1 with Oct3/4 was mediated through the POU-specific domain of Oct3/4. Ectopic expression of Dax1 inhibited Oct3/4-mediated activation of an artificial Oct3/4-responsive promoter. Expression of Dax1 in ES cells also reduced the activities of Nanog and Rex1 promoters, while knockdown of Dax1 increased these activities. Pulldown and gel shift assays revealed that the interaction of Dax1 with Oct3/4 abolished the DNA binding activity of Oct3/4. Chromatin immunoprecipitation assay results showed that Dax1 inhibited Oct3/4 binding to the promoter/enhancer regions of Oct3/4 and Nanog. Furthermore, overexpression of Dax1 resulted in ES cell differentiation. Taken together, these data suggest that Dax1, a novel molecule interacting with Oct3/4, functions as a negative regulator of Oct3/4 in ES cells.

Partially covered stent-graft implantation in rabbit aorta: a new model to investigate bioactive stent-grafts in small animals.

PURPOSE: To present a new endovascular technique for placing handmade partially covered stent-grafts in rabbit aortas that is promising for experimental study of direct gene delivery to the aortic wall. METHODS: A 7-mm-diameter Z-stent made from the inner core of a guidewire was covered with a 7-mm-diameter, 10-mm-long polyester fabric tube (2268 mL porosity). To decrease stent profile and make delivery possible through a 6-F introducer, one third of the fabric was cut away to form a partially covered polyester stent-graft. Two stent-grafts were delivered sequentially into the descending thoracic aorta of 12 male Japanese White rabbits; a third device was positioned in each aorta so that the orifices of the celiac trunk and superior mesenteric artery were not occluded. RESULTS: The implantation was successful in 10 animals. One rabbit died during the procedure due to sheath laceration of the infrarenal abdominal aorta. Another animal died within 2 days after the procedure owing to occlusion of the celiac trunk by graft fabric. At 2 weeks, the stent-grafts in the 10 surviving rabbits remained patent, and there was no migration. Gross examination of the lumens showed that both the metal stent and the polyester graft material were completely covered with thin transparent tissue, without massive thrombosis. Histological staining revealed incomplete neointima formation between the stent-graft and the aorta. Incomplete linear endothelial cells on the luminal side of the tissue ingrowth into the stent-graft were also observed. Foreign body giant cells and macrophages represented inflammatory reactions related to the graft material. CONCLUSION: Partially covered stent-grafts can be safely placed in relatively small animals and potentially used in research.

Stem cell-specific expression of Dax1 is conferred by STAT3 and Oct3/4 in embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from inner cell mass of blastocysts. An orphan nuclear receptor, Dax1, is specifically expressed in undifferentiated ES cells and plays an important role in their self-renewal. The regulatory mechanism of Dax1 expression in ES cells, however, remains unknown. In this study, we found that STAT3 and Oct3/4, essential transcription factors for ES cell self-renewal, are involved in the regulation of Dax1 expression. Suppression of either STAT3 or Oct3/4 resulted in down-regulation of Dax1. Reporter assay identified putative binding sites for these factors in the promoter/enhancer region of the Dax1 gene. Chromatin immunoprecipitation analysis suggested the in vivo association of STAT3 and Oct3/4 with the putative sites. Furthermore, gel shift assay indicated that these transcription factors directly bind to their putative binding sites. These results suggest that STAT3 and Oct3/4 control the expression of Dax1 to maintain the self-renewal of ES cells.

STAT3 and Oct-3/4 control histone modification through induction of Eed in embryonic stem cells.

Mouse embryonic stem (ES) cells can self-renew in the presence of leukemia inhibitory factor (LIF). Several essential transcription factors have been identified for the self-renewal of mouse ES cells, including STAT3, Oct-3/4, and Nanog. The molecular mechanism of ES cell self-renewal, however, is not fully understood. In the present study, we identified Eed, a core component of Polycomb repressive complex 2, as a downstream molecule of STAT3 and Oct-3/4. Artificial activation of STAT3 resulted in increased expression of Eed, whereas expression of a dominant negative mutant of STAT3 or suppression of Oct-3/4 expression led to down-regulation of Eed. Reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays revealed that STAT3 and Oct-3/4 directly bind to the promoter region of Eed, suggesting that Eed is a common target molecule of STAT3 and Oct-3/4. We also found that suppression of STAT3, Oct-3/4, or Eed causes induction of differentiation-associated genes as well as loss of Lys(27)-trimethylated histone H3 at the promoter regions of the differentiation-associated genes. Suppression of STAT3 and Oct-3/4 also resulted in the absence of Eed at the promoter regions. These results suggest that STAT3 and Oct-3/4 maintain silencing of differentiation-associated genes through up-regulation of Eed in self-renewing ES cells.

[Effect and indications of radiofrequency ablation for the treatment of obstructive sleep apnea-hypopnea syndrome].

OBJECTIVE: To explore the effect and indications of radiofrequency ablation for the treatment of obstructive sleep apnea-hypopnea syndrome. METHOD: Multilevel temperature-controlled radiofrequency therapy of soft palate, uvula, inferior turbinate, and tonsils were applied to 74 adults with obstructive sleep apnea-hypopnea syndrome (OSAHS). There were 16 mild, 23 moderate, and 35 severe cases respectively in this study. Evaluation of mucosal injury and effect of radiofrequency therapy on pain, speech and swallowing were performed early after operation. The volume of targets and length of soft palate and uvula were measured three months after operation. Polysomnography, Epworth Sleepiness Scale and Snoring Scale Score questionnaires were reevaluated six months after operation and compared with the results of pre-operation. Treatment outcome measurements were mainly based on polysomnography. RESULT: By our definition, 5 of 74 patients (6.76%) have been cured and 42 of 74 (56.76%) had improved totally. Mean Apnea-Hypopnea Index (AHI) decreased significantly and mean lowest oxygen saturation value increased significantly postoperatively (P < 0.01). The total effective rate of the patients, whose obstructive sites were all treated by radiofrequency, was remarkably higher than that of the ones, whose obstructive sites were only partly treated by radiofrequency (P < 0.01). The total effective rate of the former was 72.92%. Patients showed a significant decrease in mean score on ESS and SSS postoperatively (P < 0.01). No significant complications were observed in most patients. There were little influence on pain, speech and swallowing. The volume or length of targets decreased obviously three months after operation. CONCLUSION: Radiofrequency can reduce the volume of tissue. The short-term outcomes of radiofrequency were satisfying if obstructive sites had been all treated. This study demonstrates that the characters of radiofrequency are as follows: minimally invasive, safe, efficient, repeatable and multilevel applicable. Temperature-controlled radiofrequency therapy is a safe and effective procedure for hypertrophic infraturbinal when used separately, or as a part of a the combined approach for complex syndromes.