Chunyan Zhou

To investigate the effects of rapamycin on cardiac differentiation, murine embryonic stem cells (ESCs) were induced into cardiomyocytes by 10⁻⁴ M ascorbic acid (AA), 20 nM rapamycin alone or 0.01% solvent DMSO. We found that rapamycin alone was insufficient to initiate cardiomyogenesis. Then, the ESCs were treated with AA and rapamycin (20 nM) or AA and DMSO (0.01%) as a control. Compared with control, mouse ESCs (mESCs) treated with rapamycin (20 nM) and AA yielded a significantly higher percentage of cardiomyocytes, as confirmed by the percentage of beating embryonic bodies (EBs), the immunofluorescence and FACS analysis. Rapamycin significantly increased the expression of a panel of cardiac markers including Gata4, α-Mhc, β-Mhc, and Tnnt2. Additionally, rapamycin enhanced the expression of mesodermal and cardiac transcription factors such as Mesp1, Brachyury T, Eomes, Isl1, Gata4, Nkx2.5, Tbx5, and Mef2c. Mechanistic studies showed that rapamycin inhibits Wnt/β-catenin and Notch signaling but promotes the expression of fibroblast growth factor (Fgf8), Fgf10, and Nodal at early stage, and bone morphogenetic protein 2 (Bmp 2) at later stages. Sequential treatment of rapamycin showed that rapamycin promotes cardiac differentiation at the early and later stages. Interestingly, another mammalian target of rapamycin (mTOR) inhibitor Ku0063794 (1 µM) had similar effects on cardiomyogenesis. In conclusion, our results highlight a practical approach to generate cardiomyocytes from mESCs by rapamycin.

ISL1, a novel regulator of CCNB1 , CCNB2 and c-MYC genes, promotes gastric cancer cell proliferation and tumor growth

ISL1 is expressed in cardiac progenitor cells and plays critical roles in cardiac lineage differentiation and heart development. Cardiac progenitor cells hold great potential for clinical and translational applications. However, the mechanisms underlying ISL1 function in cardiac progenitor cells have not been fully elucidated. Here we uncover a hierarchical role of ISL1 in cardiac progenitor cells, showing that ISL1 directly regulates hundreds of potential downstream target genes that are implicated in cardiac differentiation, through an epigenetic mechanism. Specifically, ISL1 promotes the demethylation of tri-methylation of histone H3K27 (H3K27me3) at the enhancers of key downstream target genes, including Myocd and Mef2c, which are core cardiac transcription factors. ISL1 physically interacts with JMJD3, a H3K27me3 demethylase, and conditional depletion of JMJD3 leads to impaired cardiac progenitor cell differentiation, phenocopying that of ISL1 depletion. Interestingly, ISL1 is not only responsible for the recruitment of JMJD3 to specific target loci during cardiac progenitor differentiation, but also modulates its demethylase activity. In conclusion, ISL1 and JMJD3 partner to alter the cardiac epigenome, instructing gene expression changes that drive cardiac differentiation.

Runx2 is a powerful osteo-inductive factor and adipose-derived stem cells (ADSCs) are multipotent. However, it is unknown whether Runx2-overexpressing ADSCs (Runx2-ADSCs) could promote anterior cruciate ligament (ACL) reconstruction. We evaluated the effect of Runx2-ADSCs on ACL reconstruction in vitro and in vivo. mRNA expressions of osteocalcin (OCN), bone sialoprotein (BSP) and collagen I (COLI) increased over time in Runx2-ADSCs. Runx2 overexpression inhibited LPL and PPARγ mRNA expressions. Runx2 induced alkaline phosphatase activity markedly. In nude mice injected with Runx2-ADSCs, promoted bone formation was detected by X-rays 8 weeks after injection. The healing of tendon-to-bone in a rabbit model of ACL reconstruction treated with Runx2-ADSCs, fibrin glue only and an RNAi targeting Runx2, was evaluated with CT 3D reconstruction, histological analysis and biomechanical methods. CT showed a greater degree of new bone formation around the bone tunnel in the group treated with Runx2-ADSCs compared with the fibrin glue group and RNAi Runx2 group. Histology showed that treatment with Runx2-ADSCs led to a rapid and significant increase at the tendon-to-bone compared with the control groups. Biomechanical tests demonstrated higher tendon pullout strength in the Runx2-ADSCs group at early time points. The healing of the attachment in ACL reconstruction was enhanced by Runx2-ADSCs.

Islet-1 (ISL-1), a LIM-homeodomain transcription factor, has been recently found to be essential for promoting postnatal pancreatic islet proliferation. However, the detailed mechanism has not yet been elucidated. In the present study, we investigated the mechanism by which ISL-1 promotes β-cell proliferation through regulation of CyclinD1 in HIT-T15 and NIT-1 cells, as well in rat islet mass. Our results provide the evidence that ISL-1 promotes adult pancreatic islet β-cell proliferation by activating CyclinD1 transcription through cooperation with Set7/9 and PDX-1 to form an ISL-1/Set7/9/PDX-1 complex. This complex functions in an ISL-1-dependent manner, with Set7/9 functioning not only as a histone methyltransferase, which increases the histone H3K4 tri-methylation of the CyclinD1 promoter region, but also an adaptor to bridge ISL-1 and PDX-1, while PDX-1 functions as a RNA pol II binding modulator. Furthermore, the formation of the ISL-1/Set7/9/PDX-1 complex is positively associated with insulin-like growth factor-1 treatment in NIT and HIT-T15 cells in vitro, while may be negatively correlated with age in vivo.

Sepsis-induced cardiac apoptosis is one of the major pathogenic factors in myocardial dysfunction. As it enhances numerous proinflammatory factors, lipopolysaccharide (LPS) is considered the principal mediator in this pathological process. However, the detailed mechanisms involved are unclear. In this study, we attempted to explore the mechanisms involved in LPS-induced cardiomyocyte apoptosis. We found that LPS stimulation inhibited microRNA (miR)-499 expression and thereby upregulated the expression of SOX6 and PDCD4 in neonatal rat cardiomyocytes. We demonstrate that SOX6 and PDCD4 are target genes of miR-499, and they enhance LPS-induced cardiomyocyte apoptosis by activating the BCL-2 family pathway. The apoptosis process enhanced by overexpression of SOX6 or PDCD4, was rescued by the cardiac-abundant miR-499. Overexpression of miR-499 protected the cardiomyocytes against LPS-induced apoptosis. In brief, our results demonstrate the existence of a miR-499-SOX6/PDCD4-BCL-2 family pathway in cardiomyocytes in response to LPS stimulation.