BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.     

Transcriptional regulation of connective tissue growth factor by sphingosine 1-phosphate in rat cultured mesangial cells

Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-beta) via Smad activation in mesangial cells. We recently reported that sphingosine 1-phosphate (S1P) induces CTGF expression in rat cultured mesangial cells. However, the mechanism by which S1P induces CTGF expression is unknown. The present study revealed that S1P-induced CTGF expression is mediated via pertussis toxin-insensitive pathways, which are involved in the activation of small GTPases of the Rho family and protein kinase C. We also showed by luciferase reporter assays and chromatin immunoprecipitation that S1P induces CTGF expression via Smad activation as TGF-beta does.     

Transcriptional control of the human aldehyde dehydrogenase 2 promoter by hepatocyte nuclear factor 4: inhibition by cyclic AMP and COUP transcription factors.

An important regulatory element (designated FP330-3') of the ALDH2 promoter mediates activation by hepatocyte nuclear factor 4 (HNF4). This activation of promoter constructs containing this element by HNF4 was reduced by nearly half by 8-Br-cAMP in H4IIEC3 cells, an effect that was blocked by inhibitors of protein kinase A (PKA). Cotransfection assays showed that COUP-TF I, ARP-1, or PPARdelta suppressed the ability of HNF4 to activate the reporter. The repression was potentiated by 8-Br-cAMP. Electrophoretic mobility shift assays revealed that treatment of hepatoma cells or cultured rat hepatocytes with 1 mM 8-Br-cAMP or glucagon reduced binding of FP330-3' by HNF4 by half. In vitro phosphorylation of HNF4 by PKA decreased binding to FP330-3'. Fasting reduced the ALDH2 protein level in liver and kidney, two tissues expressing HNF4, but not heart. These data suggest that ALDH2 expression can be suppressed by cAMP, most likely through phosphorylation of HNF4 by PKA, and this may account for the reduction in enzyme protein during fasting.     

Differential regulation of HSP70 expression by the JNK kinases SEK1 and MKK7 in mouse embryonic stem cells treated with cadmium

JNK, a member of the mitogen-activated protein kinases (MAPKs), is activated by the MAPK kinases SEK1 and MKK7 in response to environmental stresses. In the present study, the effects of CdCl2 treatment on MAPK phosphorylation and HSP70 expression were examined in mouse embryonic stem (ES) cells lacking the sek1 gene, the mkk7 gene, or both. Following CdCl2 exposure, the phosphorylation of JNK, p38, and ERK was suppressed in sek1-/- mkk7-/- cells. When sek1-/- or mkk7-/- cells were treated with CdCl2, JNK phosphorylation, but not the phosphorylation of either p38 or ERK, was markedly reduced, while a weak reduction in p38 phosphorylation was observed in sek1-/- cells. Thus, both SEK1 and MKK7 are required for JNK phosphorylation, whereas their role in p38 and ERK phosphorylation could overlap with that of another kinase. We also observed that CdCl2-induced HSP70 expression was abolished in sek1-/- mkk7-/- cells, was reduced in sek1-/- cells, and was enhanced in mkk7-/- cells. Similarly, the phosphorylation of heat shock factor 1 (HSF1) was decreased in sek1-/- mkk7-/- and sek1-/- cells, but was increased in mkk7-/- cells. Transfection with siRNA specific for JNK1, JNK2, p38, ERK1, or ERK2 suppressed CdCl2-induced HSP70 expression. In contrast, silencing of p38 or p38 resulted in further accumulation of HSP70 protein. These results suggest that HSP70 expression is up-regulated by SEK1 and down-regulated by MKK7 through distinct MAPK isoforms in mouse ES cells treated with CdCl2.     

Ginsenoside Rg1 facilitates neural differentiation of mouse embryonic stem cells via GR-dependent signaling pathway

Ginsenoside Rg1, a steroidal saponin of high abundance in ginseng, possesses the neuroprotective effects. In this study, we tried to explore the effect of Rg1 on promoting differentiation of mouse embryonic stem (ES) cells towards the neuronal lineage and its potential role involved in glucocorticoid receptor (GR) activation. Rg1 treatment induced a remarkable increase in the population of neuron-like cells in a time-dependent manner. More than 80% of Rg1-treated embryoid bodies (EBs) differentiated into neuron-like cells on d 8 + 10. Furthermore, the gradually increased protein expression of neurofilament (NEFM) and β-tubulin III (a neuronal specific protein) was determined. GR expression gradually increased during the differentiation course. RU486, an antagonist of GR, could efficiently block the neurogenesis-promoting activity of Rg1. On the other side, Rg1 stimulated the phosphorylation of ERK1/2 and Akt at different time points through GR activation-dependent mechanisms. Treatment of both U0126 (an inhibitor of MEK) and LY294002 (an inhibitor of PI3 K), hampered the neuronal differentiation induced by Rg1. Meantime, U0126 further decreased Rg1-induced p-Akt expression. In conclusion, Rg1 possesses the effects on inducing differentiation of mouse ES cells into neurons in vitro via the GR-MEK-ERK1/2-PI3 K-Akt signaling pathway.