PPAR-beta facilitating maturation of hepatic-like tissue derived from mouse embryonic stem cells accompanied by mitochondriogenesis and membrane potential retention.

Relatively little is known about mitochondria metabolism in differentiating embryonic stem (ES) cells. Present research focused on several elements of cellular energy metabolism in hepatic-like tissue derived from mouse ES cells. We demonstrated that mitochondrial location patterns and mitochondrial membrane potential (DeltaPsi(m)) existed in subsequent differentiation of the tissue. Mitochondriogenesis appeared at the early stage and kept a normal DeltaPsi(m) in differentiated mature hepatocytes. Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) expression was transitorily increased at the beginning, and kept a relatively low level later, which accompanied by expression of PPAR-gamma coactivator (PGC)-1alpha, a master regulator of mitochondrial biogenesis. PPAR-beta expression showed robust up-regulation in the late differentiation course. Enhanced co-expressions of PPAR-beta and albumin with catalysis of UDP-glucuronosyltransferases (UGTs) were observed at mature stage. While PPAR-gamma expression changed little before and after differentiation. Mitochondriogenesis could be accelerated by PPAR-alpha specific agonist WY14643 and abolished by its antagonist GW6471 at the early stage. Neither of them affected mitochondrial DeltaPsi(m) and albumin generation in the differentiated hepatocytes. Furthermore, maturation of hepatic-like tissue and mitochondriogenesis in hepatocyte could be efficiently stimulated by PPAR-beta specific agonist L165041 and abolished by PPAR-beta specific antagonist GSK0660, but not affected by PPAR-gamma specific agonist GW1929. In conclusion, the derived hepatic tissue morphologically possessed cellular energy metabolism features. PPAR-alpha seemed only necessary for early mitochondriogenesis, while less important for DeltaPsi(m) retention in the mature tissue derived. The stimulation of PPAR-beta but not -gamma enhanced hepatogenesis, hepatocytes maturation, and mitochondriogenesis. PPAR-beta took an important role in cellular energy metabolism of hepatogenesis.

Involvement of NF-kappaB and AP-1 activation in icariin promoted cardiac differentiation of mouse embryonic stem cells.

Icariin has been reported to facilitate the differentiation of mouse embryonic stem (ES) cells into cardiomyocytes; however, the mechanism on cardiomyogenic cell lineage differentiation has not been fully elucidated yet. In the present studies, an underlying signaling network including p38, extracellular signal-regulated kinase 1, 2 (ERK1, 2), nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) transcription factors c-jun and c-fos was assumed in icariin induced cardiomyogenesis. Icariin rapidly activated p38 and ERK1, 2 in embryoid bodies, treatment with p38 antagonist 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) or ERK1, 2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126) significantly abolished icariin induced cardiac commitment, MEF2C gene expression and nuclear translocation, as well as cardiac-specific protein alpha-actinin expression, indicating that p38 and ERK1, 2 are specifically involved in icariin stimulated cardiomyogenic cell lineage differentiation of ES cells. Further, IkappaBalpha phosphorylation and NF-kappaB p65 translocation to the nucleus appeared rapidly when embryoid bodies exposed to icariin, and the expression of IkappaBalpha or NF-kappaB p65 in cytoplasm was decreased concomitantly. Moreover, icariin increased c-jun and c-fos mRNA and protein expression. Either SB203580 or U0126 displayed inhibitory effect on icariin induced NF-kappaB and AP-1 activation. It could be concluded that p38 and ERK1, 2 are activated in a coordinated manner, which in turn contribute to NF-kappaB and AP-1 activation in icariin induced cardiomyogenic cell lineage differentiation of mouse ES cells.

Reactive oxygen species involved in prenylflavonoids, icariin and icaritin, initiating cardiac differentiation of mouse embryonic stem cells.

The significant promoting effects of some prenylflavonoids on cardiac differentiation of mouse embryonic stem (ES) cells via reactive oxygen species (ROS) signaling pathway were investigated. The most effective differentiation was facilitated by icariin (ICA), followed by icaritin (ICT), while desmethylicaritin (DICT) displayed the weakest but still significant inducible effect. Contrarily, DICT demonstrated the strongest anti-oxidative activity while ICA displayed only little in vitro, which was well matched with the hydroxyl (OH) numbers and the positions in the molecular structures. Therefore, ROS signaling cascades were assumed to be involved in prenylflavonoids induced cardiomyogenesis. Treatment with ICA, intracellular ROS in embryoid bodies was rapidly elevated, which was abolished by the NADPH-oxidase inhibitor apocynin; elimination of intracellular ROS by vitamin E or pyrrolidine dithiocarbamate (PDTC) inhibited ICA induced cardiomyogenesis; ROS-sensitive extracellular-regulated kinase 1, 2 (ERK1, 2) and p38 activation were further observed, the cardiomyogenesis was significantly inhibited in the presence of ERK1, 2 or p38 inhibitor U0126 or SB203580, indicating the roles of NADPH-ROS-MAPKs signaling cascades in prenylflavonoids induced cardiac differentiation. There was no difference in Nox4 NADPH oxidase expression between ICA and ICT treatments, however, ROS concentration in EBs after ICT administration was lower than that after ICA treatment, followed by less activation of ERK1, 2, and p38. These results revealed that the significant promoting effects of prenylflavonoids on cardiac differentiation was at least partly via ROS signaling cascades, and the facilitating abilities preferentially based on the nature of prenylflavonoids themselves, but anti-oxidative activity determined by the OH numbers and the positions in the structures do influence the cardiomyogenesis in vitro.