miR-200c and GATA binding protein 4 regulate human embryonic stem cell renewal and differentiation.

Human embryonic stem cells (hESCs) are functionally unique for their self-renewal ability and pluripotency, but the molecular mechanisms giving rise to these properties are not fully understood. hESCs can differentiate into embryoid bodies (EBs) containing ectoderm, mesoderm, and endoderm. In the miR-200 family, miR-200c was especially enriched in undifferentiated hESCs and significantly downregulated in EBs. The knockdown of the miR-200c in hESCs downregulated Nanog expression, upregulated GATA binding protein 4 (GATA4) expression, and induced hESC apoptosis. The knockdown of GATA4 rescued hESC apoptosis induced by downregulation of miR-200c. miR-200c directly targeted the 3'-untranslated region of GATA4. Interestingly, the downregulation of GATA4 significantly inhibited EB formation in hESCs. Overexpression of miR-200c inhibited EB formation and repressed the expression of ectoderm, endoderm, and mesoderm markers, which could partially be rescued by ectopic expression of GATA4. Fibroblast growth factor (FGF) and activin A/nodal can sustain hESC renewal in the absence of feeder layer. Inhibition of transforming growth factor-ß (TGF-ß[Symbol: see text])/activin A/nodal signaling by SB431542 treatment downregulated the expression of miR-200c. Overexpression of miR-200c partially rescued the expression of Nanog/phospho-Smad2 that was downregulated by SB431542 treatment. Our observations have uncovered novel functions of miR-200c and GATA4 in regulating hESC renewal and differentiation.

Defective phosphatidylinositol 3-kinase signaling in central control of cardiovascular effects in the nucleus tractus solitarii of spontaneously hypertensive rats.

Recently we have shown functional involvement of the phosphatidylinositol 3-kinase (PI3K)-Akt-nitric oxide synthase (NOS) signaling pathway in central control of cardiovascular effects in the nucleus tractus solitarii (NTS) of normotensive Wistar-Kyoto (WKY) rats. In this study we determined whether PI3K/Akt signaling was defective in spontaneously hypertensive rats (SHR). WKY rats and SHR were anesthetized with urethane. Mean blood pressure (MBP) and heart rate (HR) were monitored intra-arterially. Unilateral microinjection (60 nL) of insulin (100 IU/mL) into the NTS produced prominent depressor and bradycardic effects in 8- and 16-week-old normotensive WKY and 8-week-old SHR. However, no significant cardiovascular effects were found in 16-week-old SHR after insulin injection. Furthermore, pretreatment with PI3K inhibitor LY294002 and NOS inhibitor L-NAME into the NTS attenuated the cardiovascular response evoked by insulin in WKY and 8-week-old SHR but not in 16-week-old SHR. Unilateral microinjection of 1 mmol/L of PI(3,4,5)P(3) (phosphatidylinositol 3,4,5-triphosphate), a phospholipids second messenger produced by PI3K, into the NTS produced prominent depressor and bradycardic effects in 8- or 16-week-old WKY rats as well as 8-week-old SHR but not in 16-week-old SHR. Western blot analysis showed no significant increase in Akt phosphorylation in 8-week-old pre-hypertensive SHR after insulin injection. Similar results were also found in hypertensive 16-week-old SHR. Our results indicate that the Akt-independent signaling pathway is involved in NOS activation to regulate cardiovascular effects in the NTS of 8-week-old pre-hypertensive SHR. Both Akt-dependent and Akt-independent signaling pathways are defective in hypertensive 16-week-old SHR.

In situ Akt phosphorylation in the nucleus tractus solitarii is involved in central control of blood pressure and heart rate.

BACKGROUND: Previously, we have shown that nitric oxide (NO) plays a significant role in central cardiovascular regulation and modulates the baroreflex in the nucleus tractus solitarii (NTS) of rats. NO production is mediated by activation of NO synthase (NOS). Insulin signaling was involved in controlling peripheral blood pressure via the activation of endothelial NOS. Here, we investigated whether the insulin signal transduction pathway is involved in controlling central cardiovascular effects. METHODS AND RESULTS: Insulin was injected into NTS of urethane-anesthetized male Wistar-Kyoto (WKY) rats. Unilateral microinjection (60 nL) of insulin (100 IU/mL) into the NTS produced prominent depressor and bradycardic effects in 8- and 16-week-old WKY rats. In addition, pretreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the NOS inhibitor L-NAME into the NTS caused attenuation of the cardiovascular response evoked by insulin in either 8- or 16-week-old WKY rats. Western blot analysis showed a significant increase (2.6+/-0.4-fold; P<0.05) in Akt phosphorylation after insulin injection, whereas LY294002 abolished the insulin-induced effects. In situ Akt phosphorylation was found in NTS by immunohistochemistry analysis after injection of insulin. This in situ Akt phosphorylation was abolished significantly after injection of LY294002. CONCLUSIONS: Take together, these results suggest that the insulin-PI3K-Akt-NOS signaling pathway may play a significant role in central cardiovascular regulation.