ngsong Li

Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation.

Neural stem cells (NSCs) constitute the cellular basis for embryonic brain development and neurogenesis. The process is regulated by NSC niche including neighbor cells such as vascular and glial cells. Since both vascular and glial cells secrete vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), we assessed the effect of VEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonic stem cells. VEGF alone did not have any significant effect. When bFGF was added, however, VEGF stimulated NSC proliferation in a dose-dependent manner, and this stimulation was inhibited by ZM323881, a VEGF receptor (Flk-1)-specific inhibitor. Interestingly, ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF, suggesting that VEGF autocrine plays a role in the proliferation of NSCs. The stimulatory effect of VEGF on NSC proliferation depends on bFGF, which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphorylation of ERK1/2. Collectively, this study may provide insight into the mechanisms by which microenvironmental niche signals regulate NSCs.

The Egf Receptor-Sox2-Egf Receptor Feedback Loop Positively Regulates the Self-Renewal of Neural Precursor Cells†‡§

The transcriptional factor Sox2 and epidermal growth factor receptor (Egfr)‐mediated signaling are both required for self‐renewal of neural precursor cells (NPCs). However, the mechanism by which these factors coordinately regulate this process is largely unknown. Here we show that Egfr‐mediated signaling promotes Sox2 expression, which in turn binds to the Egfr promoter and directly upregulates Egfr expression. Knockdown of Sox2 by RNA interference downregulates Egfr expression and attenuates colony formation of NPCs, whereas overexpression of Sox2 elevates Egfr expression and promotes NPC self‐renewal. Moreover, the effect of Sox2 on NPC self‐renewal is completely inhibited by AG1478, a specific inhibitor for Egfr; it is also inhibited by LY294002 and U0126, selective antagonists for phosphatidylinositol 3‐kinase (PI3K) and extracellular signal‐regulated kinase (Erk1/2), respectively. Collectively, we conclude that NPC self‐renewal is enhanced through a novel cellular feedback loop with mutual regulation of Egfr and Sox2. STEM CELLS 2010;28:279–286

Paired box 6 (PAX6) regulates glucose metabolism via proinsulin processing mediated by prohormone convertase 1/3 (PC1/3)

Aims/hypothesisHuman patients with aniridia caused by heterozygous PAX6 mutations display abnormal glucose metabolism, but the underlying molecular mechanism is largely unknown. Disturbed islet architecture has been proposed as the reason why mice with complete inactivation of paired box 6 (PAX6) in the pancreas develop diabetes. This is not, however, the case in human aniridia patients with heterozygous PAX6 deficiency and no apparent defects in pancreatic development. We investigated the molecular mechanism underlying the development of abnormal glucose metabolism in these patients.MethodsA human aniridia pedigree with a PAX6 R240Stop mutation was examined for abnormal glucose metabolism using an OGTT. The underlying mechanism was further investigated using Pax6 R266Stop mutant small-eye mice, which also have abnormal glucose metabolism similar to that in PAX6 R240Stop mutation human aniridia patients.ResultsPaired box 6 (PAX6) deficiency, both in aniridia patients with a heterozygous PAX6 R240Stop mutation and in mice with a heterozygous Pax6 R266Stop mutation, causes defective proinsulin processing and abnormal glucose metabolism. PAX6 can bind to the promoter and directly upregulate production of prohormone convertase (PC)1/3, an enzyme essential for conversion of proinsulin to insulin. Pax6 mutations lead to PC1/3 deficiency, resulting in defective proinsulin processing and abnormal glucose metabolism.Conclusions/interpretationThis study indicates a novel function for PAX6 in the regulation of proinsulin processing and glucose metabolism via modulation of PC1/3 production. It also provides an insight into the abnormal glucose metabolism caused by heterozygous PAX6 mutations in humans and mice.

Human embryonic germ cells isolation from early stages of post-implantation embryos

Human embryonic germ (hEG) cell is a very important alternative pluripotent stem cell resource. We describe the derivation of hEG cells from human embryonic fetal gonads over 6–8 weeks postconception. A large number of EG-like cell clumps were obtained at passage 1 and thus facilitated the following routine culture when the donor tissues were trypsinized with gentle pipetting and plated on feeder layer cells in the initial culture. Eight diploid hEG cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent stem cells. Human EG cells expressed transcription factor Oct4, a marker of pluripotency in mouse EG cells, at a high and steady level. Expression of markers indicative of differentiation along the three germ lineages was also observed in EBs. High level of alkaline phosphatase activity was shown in EG cells. These encouraging findings provide a starting point for potential applicability of hEG cells.

Sox2 protects neural stem cells from apoptosis via up-regulating survivin expression.

The transcription factor Sox2 [SRY (sex-determining region Y)-box 2] is essential for the regulation of self-renewal and homoeostasis of NSCs (neural stem cells) during brain development. However, the downstream targets of Sox2 and its underlying molecular mechanism are largely unknown. In the present study, we found that Sox2 directly up-regulates the expression of survivin, which inhibits the mitochondria-dependent apoptotic pathway in NSCs. Although overexpression of Sox2 elevates survivin expression, knockdown of Sox2 results in a decrease in survivin expression, thereby initiating the mitochondria-dependent apoptosis related to caspase 9 activation. Furthermore, cell apoptosis owing to knockdown of Sox2 can be rescued by ectopically expressing survivin in NSCs as well as in the mouse brain, as demonstrated by an in utero-injection approach. In short, we have found a novel Sox2/survivin pathway that regulates NSC survival and homoeostasis, thus revealing a new mechanism of brain development, neurological degeneration and such aging-related disorders.

Lysophosphatidic acid accelerates lung fibrosis by inducing differentiation of mesenchymal stem cells into myofibroblasts

Lung fibrosis is characterized by vascular leakage and myofibroblast recruitment, and both phenomena are mediated by lysophosphatidic acid (LPA) via its type‐1 receptor (LPA1). Following lung damage, the accumulated myofibroblasts activate and secrete excessive extracellular matrix (ECM), and form fibrotic foci. Studies have shown that bone marrow‐derived cells are an important source of myofibroblasts in the fibrotic organ. However, the type of cells in the bone marrow contributing predominantly to the myofibroblasts and the involvement of LPA‐LPA1 signalling in this is yet unclear. Using a bleomycin‐induced mouse lung‐fibrosis model with an enhanced green fluorescent protein (EGFP) transgenic mouse bone marrow replacement, we first demonstrated that bone marrow derived‐mesenchymal stem cells (BMSCs) migrated markedly to the bleomycin‐injured lung. The migrated BMSC contributed significantly to α‐smooth muscle actin (α‐SMA)‐positive myofibroblasts. By transplantation of GFP‐labelled human BMSC (hBMSC) or EGFP transgenic mouse BMSC (mBMSC), we further showed that BMSC might be involved in lung fibrosis in severe combined immune deficiency (SCID)/Beige mice induced by bleomycin. In addition, using quantitative‐RT‐PCR, western blot, Sircol collagen assay and migration assay, we determined the underlying mechanism was LPA‐induced BMSC differentiation into myofibroblast and the secretion of ECM via LPA1. By employing a novel LPA1 antagonist, Antalpa1, we then showed that Antalpa1 could attenuate lung fibrosis by inhibiting both BMSC differentiation into myofibroblast and the secretion of ECM. Collectively, the above findings not only further validate LPA1 as a drug target in the treatment of pulmonary fibrosis but also elucidate a novel pathway in which BMSCs contribute to the pathologic process.

Pax6 directly modulate Sox2 expression in the neural progenitor cells.

Pax6 is a key regulator in the neuronal fate determination as well as the proliferation of neural stem cells, but the mechanisms are still unknown. Our study shows that Pax6 regulate the proliferation of neural progenitor cells of cortical subventricular zone, through direct modulation of the Sox2 expression during the late developmental stage in mice. We found a dramatic decrease in the number of Sox2+ neural progenitor cells in the subventricular zone of E18.5 Pax6−/− mice. We confirmed that Pax6 could bind to the Sox2 promoter by chromatin immunoprecipitation assay and activate Sox2 expression by a luciferase reporter gene assay. Moreover, neural progenitors isolated from the Pax6−/− embryos showed a decreased neurosphere formation as well as proliferation.

Pax6 Directly Down-Regulates Pcsk1n Expression Thereby Regulating PC1/3 Dependent Proinsulin Processing

Background Heterozygous paired box6 (Pax6) mutations lead to abnormal glucose metabolism in mice older than 6 months as well as in human beings. Our previous study found that Pax6 deficiency caused down-expression of prohormone convertase 1/3 (Pcsk1), resulting in defective proinsulin processing. As a protein cleaving enzyme, in addition to its expression, the activity of PC1/3 is closely related to its function. We therefore hypothesize that Pax6 mutation alters the activity of PC1/3, which affects proinsulin processing. Methodology/Principal Findings Using quantitative RT-PCR, western blot and enzyme assay, we found that PC1/3 C-terminal cleavage and its activity were compromised in Pax6 R266Stop mutant mice, and the expression of Pcsk1n, a potent inhibitor of PC1/3, was elevated by Pax6 deficiency in the mutant mice and MIN6 cells. We confirmed the effect of proSAAS, the protein encoded by Pcsk1n, on PC1/3 C-terminal cleavage and its activity by Pcsk1n RNAi in MIN6 cells. Furthermore, by luciferase-reporter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assay, we revealed that Pax6 bound to Pcsk1n promoter and directly down-regulated its expression. Finally, by co-transfecting Pax6 siRNA with Pcsk1n siRNA, we showed that Pax6 knock-down inhibited proinsulin processing and that this effect could be rescued by proSAAS down-regulation. These findings confirm that Pax6 regulates proinsulin processing partially through proSAAS-mediated PC1/3 processing and activity. Conclusions/Significance Collectively, the above experiments demonstrate that Pax6 can directly down-regulate Pcsk1n expression, which negatively affects PC1/3 C-terminal cleavage and activity and subsequently participates in proinsulin processing. We identified proSAAS as a novel down-regulated target of Pax6 in the regulation of glucose metabolism. This study also provides a complete molecular mechanism for the Pax6 deficiency-caused diabetes.

Oct4 is expressed in Nestin-positive cells as a marker for pancreatic endocrine progenitor

There are abundant progenitor cells in the developing pancreas, but molecular markers for these cells are lacking. Octamer-binding transcription factor-4 (Oct4) is an important transcription factor for keeping the features of self-renewal and pluripotency of embryonic stem cells. It’s well known that Oct4, as a totipotent stem cells marker, just is expressed in totipotent stem cells. In the present study, we collected ten human fetal pancreases, and found that Oct4 mRNA and protein were expressed in human fetal pancreas samples by RT-PCR, western blot and immunohistochemistry assays. Using double-staining, we demonstrated that Oct4 was not co-expressed with Chromogranin A (a peptide expressed in endocrine cells), but partially co-expressed with Ngn3 (a transcription factor expressed in pancreatic endocrine precursor cells) and Nestin (a intermediate filament, Nestin-positive cells isolated from islets can be induced to express insulin) in human fetal pancreases. Indeed, we prepared Nestin-positive cells from human fetal pancreas by cell selection, and found that these cells expressed Oct4 and Ngn3. The Nestin-positive cells displayed a rapid duplication and could differentiate into osteoblasts, fat and endocrine cells in vitro. These results indicated that the Nestin-positive cells in the fetal age should be pancreatic progenitor cells. Overall, our study suggested that Oct4 was a marker for pancreatic endocrine progenitor.

Novel Mutations of Cathepsin C Gene in Two Chinese Patients with Papillon-Lefèvre Syndrome

Papillon-Lefèvre syndrome (PLS) is an inherited human disease characterized by early-onset periodontitis and palmoplantar hyperkeratosis. Mutations of the lysosomal protease cathepsin C (CTSC) gene have been shown to be the genetic cause of Papillon-Lefèvre syndrome. There are several case reports in China, while there has been no study on the genetic analysis of PLS. We studied two Chinese patients carrying Papillon-Lefèvre syndrome and showing premature tooth loss and palmoplantar hyperkeratosis. Mutation screening and sequence analysis of the CTSC gene revealed a compound heterozygous mutation (c.415 G>A and c.778 T>C) in one patient, and two novel compound heterozygous mutations (c.851G>A and c.112delCCTG) in the other patient. Our novel discovery indicates that the phenotypes observed in these two patients are due to the CTSC gene mutation.