Yinan Liu

WNT signaling promotes Nkx2.5 expression and early cardiomyogenesis via downregulation of Hdac1.

The cardiac transcription factor NKX2.5 plays a crucial role in cardiomyogenesis, but its mechanism of regulation is still unclear. Recently, epigenetic regulation has become increasingly recognized as important in differentiation and development. In this study, we used P19CL6 cells to investigate the regulation of Nkx2.5 expression by methylation and acetylation during cardiomyocyte differentiation. During the early stage of differentiation, Nkx2.5 expression was upregulated, but the methylation status of the Nkx2.5 promoter did not undergo significant change; while the acetylation levels of histones H3 and H4 were increased, accompanied by a significant reduction in Hdac1 expression. Suppression of Hdac1 activity stimulated cardiac differentiation accompanied by increased expression of cardiac-specific genes and cell cycle arrest. Overexpression of Hdac1 inhibited cardiomyocyte formation and downregulated the expressions of Gata4 and Nkx2.5. Mimicking induction of the WNT pathway inhibited Hdac1 expression with upregulated Nkx2.5 expression. WNT3a and WNT3 downregulated the expression of Hdac1, contrary to the effect of SFRP2 and GSK3beta. Cotransfection of beta-catenin and Lef1 significantly downregulated the expression of Hdac1. Our data suggest that WNT signaling pathway plays important roles in the regulation of Hdac1 during the early stage of cardiomyocyte differentiation and that the downregulation of Hdac1 promotes cardiac differentiation.

ISL1 Promotes Pancreatic Islet Cell Proliferation

Background Islet 1 (ISL1), a LIM-homeodomain transcription factor is essential for promoting pancreatic islets proliferation and maintaining endocrine cells survival in embryonic and postnatal pancreatic islets. However, how ISL1 exerts the role in adult islets is, to date, not clear. Methodology/Principal Findings Our results show that ISL1 expression was up-regulated at the mRNA level both in cultured pancreatic cells undergoing glucose oxidase stimulation as well in type 1 and type 2 diabetes mouse models. The knockdown of ISL1 expression increased the apoptosis level of HIT-T15 pancreatic islet cells. Using HIT-T15 and primary adult islet cells as cell models, we show that ISL1 promoted adult pancreatic islet cell proliferation with increased c-Myc and CyclinD1 transcription, while knockdown of ISL1 increased the proportion of cells in G1 phase and decreased the proportion of cells in G2/M and S phases. Further investigation shows that ISL1 activated both c-Myc and CyclinD1 transcription through direct binding on their promoters. Conclusions/Significance ISL1 promoted adult pancreatic islet cell proliferation and probably by activating c-Myc and CyclinD1 transcription through direct binding on their promoters. Our findings extend the knowledge about the crucial role of ISL1 in maintaining mature islet cells homeostasis. Our results also provide insights into the new regulation relationships between ISL1 and other growth factors.

β-Catenin/TCF/LEF1 can directly regulate phenylephrine-induced cell hypertrophy and Anf transcription in cardiomyocytes

beta-Catenin/TCF/LEF1 signaling is implicated in cardiac hypertrophy. We demonstrate that knockdown of beta-catenin attenuates phenylephrine (PE)-induced cardiomyocyte hypertrophy and the up-regulation of the fetal gene Anf. We explore the mechanism through which beta-catenin regulates Anf expression and find a consensus binding sequence on the Anf promoter for TCF/LEF1 family members. LEF1 binds directly to the Anf promoter via this sequence, which shows functional significance, and PE stimulation enhances recruitment of beta-catenin onto the Anf promoter. Thus, we document a direct positive role of beta-catenin on PE-induced cardiomyocyte hypertrophy and identify a new target gene for beta-catenin/TCF/LEF1.

Isolation and characterization of epithelial progenitor cells from human fetal liver

Aim:  Hepatic progenitor cells can serve as an alternative source of hepatocytes for the treatment of liver diseases.

Timely inhibition of Notch signaling by DAPT promotes cardiac differentiation of murine pluripotent stem cells.

The Notch signaling pathway plays versatile roles during heart development. However, there is contradictory evidence that Notch pathway either facilitates or impairs cardiomyogenesis in vitro. In this study, we developed iPSCs by reprogramming of murine fibroblasts with GFP expression governed by Oct4 promoter, and identified an effective strategy to enhance cardiac differentiation through timely modulation of Notch signaling. The Notch inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) alone drove the iPSCs to a neuronal fate. After mesoderm induction of embryoid bodies initiated by ascorbic acid (AA), the subsequent treatment of DAPT accelerated the generation of spontaneously beating cardiomyocytes. The timed synergy of AA and DAPT yielded an optimal efficiency of cardiac differentiation. Mechanistic studies showed that Notch pathway plays a biphasic role in cardiomyogenesis. It favors the early–stage cardiac differentiation, but exerts negative effects on the late-stage differentiation. Therefore, DAPT administration at the late stage enforced the inhibition of endogenous Notch activity, thereby enhancing cardiomyogenesis. In parallel, DAPT dramatically augmented the expression of Wnt3a, Wnt11, BMP2, and BMP4. In conclusion, our results highlight a practicable approach to generate cardiomyocytes from iPSCs based on the stage-specific biphasic roles of Notch signaling in cardiomyogenesis.

A Minority Subpopulation of CD133+/EGFRvIII+/EGFR− Cells Acquires Stemness and Contributes to Gefitinib Resistance

To study the contribution of epidermal growth factor receptor variant III (EGFRvIII) to glioblastoma multiforme (GBM) stemness and gefitinib resistance.

POU homeodomain protein OCT1 modulates islet 1 expression during cardiac differentiation of P19CL6 cells.

Islet 1 (ISL1), a marker of cardiac progenitors, plays a crucial role in cardiogenesis. However, the precise mechanism underlying the activation of its expression is not fully understood. Using the cardiac differentiation model of P19CL6 cells, we show that POU homeodomain protein, OCT1, modulates Isl1 expression in the process of cardiac differentiation. Oct1 knock-down resulted in reduction of Isl1 expression and downregulated mesodermal, cardiac-specific, and signal pathway gene expression. Additionally, the octamer motif located in the proximal region of Isl1 promoter is essential to Isl1 transcriptional activation. Mutation of this motif remarkably decreased Isl1 transcription. Although both OCT1 and OCT4 bound to this motif, it was OCT1 rather than OCT4 that modulated Isl1 expression. Furthermore, the correlation of OCT1 in regulation of Isl1 was revealed by in situ hybridization in early embryos. Collectively, our data highlight a novel role of OCT1 in the regulation of Isl1 expression.

C/EBPβ promotes angiogenesis through secretion of IL-6, which is inhibited by genistein, in EGFRvIII-positive glioblastoma.

To study the mechanisms underlying the IL‐6‐promoted angiogenic microenvironment in EGFRvIII‐positive glioblastoma, VEGF expression in EGFRvIII‐positive/negative tumors was determined by optical molecular imaging. Next, the HUVEC tube formation assay, Western blot, qPCR, RNA silencing, chromatin immunoprecipitation, luciferase reporter and ELISA assays were performed to examine the role of IL‐6 and C/EBPβ in the formation of the angiogenic microenvironment in EGFRvIII‐positive tumors. Finally, in vitro and in vivo genistein treatment experiments were conducted to challenge the interaction between the IL‐6 promoter and C/EBPβ. Optical imaging revealed greater VEGF expression in EGFRvIII‐positive tumor‐bearing mice, suggesting an angiogenic microenvironment. In vitro experiments demonstrated that C/EBPβ‐mediated regulation of IL‐6 was indispensable for maintenance of this angiogenic microenvironment. In contrast, genistein‐mediated upregulation of CHOP impeded C/EBPβ interaction with the IL‐6 promoter, thus disturbing the angiogenic microenvironment. This more malignant microenvironment in EGFRvIII glioblastoma is generated, at least in part, by greater VEGF, IL‐6 and C/EBPβ expression. Interaction of C/EBPβ with the IL‐6 promoter maintains this angiogenic microenvironment, while disturbance of this dynamically balanced interaction inhibits EGFRvIII tumor proliferation by reducing both VEGF and IL‐6 expression.

Knock out CD44 in reprogrammed liver cancer cell C3A increases CSCs stemness and promotes differentiation

CD44 is a widely known cancer stem cells marker in various cancers and validated to function in tumor growth, survival and tumor metastasis. In this study, we first established C3A-derived liver cancer stem cells by OSKM method [OCT4, SOX2, KLF4, and c-MYC], termed C3A-induced cancer stem cells (C3A-iCSCs) which acquired self-renewal and stemness abilities. Then we found CD44 was positive in C3A-iCSCs and mainly located in cell nuclear. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) results showed nuclear CD44 combined promoter regions of c-MYC and SOX2. These results suggested that CD44 participated in C3A-iCSCs transcriptional regulation. To explore CD44 overall influence in liver cancer stem cells, CD44 was knocked out in C3A-iCSCs using CRISPR/Cas9 technology. Our results showed a dramatic increase in the expression of stem cell markers OCT4, SOX2 and NANOG in CD44− C3A-iCSCs compared with that in CD44+ C3A-iCSCs. Tumor derived from CD44− C3A-iCSCs also displayed well-differentiated tumor cells compared to CD44+ C3A-iCSCs, which suggested CD44− C3A-iCSCs derived tumor cells exhibited lower malignant degree. Our data indicated nuclear CD44 in liver cancer stem cells is responsible for the poorly differentiated highly malignant tumor cells by maintenance of low stemness state.

Angiopoietin1 inhibits mast cell activation and protects against anaphylaxis.

Since morbidity and mortality rates of anaphylaxis diseases have been increasing year by year, how to prevent and manage these diseases effectively has become an important issue. Mast cells play a central regulatory role in allergic diseases. Angiopoietin1 (Ang-1) exhibits anti-inflammatory properties by inhibiting vascular permeability, leukocyte migration and cytokine production. However, Ang-1s function in mast cell activation and anaphylaxis diseases is unknown. The results of our study suggest that Ang-1 decreased lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production of mast cells by suppressing IκB phosphorylation and NF-κB nuclear translocation. Ang-1 also strongly inhibited compound 48/80 induced and FcεRI-mediated mast cells degranulation by decreasing intracellular calcium levels in vitro. In vivo lentivirus-mediated delivery of Ang-1 in mice exhibited alleviated leakage in IgE-dependent passive cutaneous anaphylaxis (PCA). Furthermore, exogenous Ang-1 intervention treatment prevented mice from compound 48/80-induced mesentery mast cell degranulation, attenuated increases in pro-inflammatory cytokines, relieved lung injury, and improved survival in anaphylaxis shock. The results of our study reveal, for the first time, the important role of Ang-1 in the activation of mast cells, and identify a therapeutic effect of Ang-1 on anaphylaxis diseases.