ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells.

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.

SOX6 and PDCD4 enhance cardiomyocyte apoptosis through LPS-induced miR-499 inhibition.

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.

ISL-1 is overexpressed in non-Hodgkin lymphoma and promotes lymphoma cell proliferation by forming a p-STAT3/p-c-Jun/ISL-1 complex.

BACKGROUND: Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, is essential for the heart, motor neuron and pancreas development. Recently, ISL-1 has been found in some types of human cancers. However, how ISL-1 exerts the role in tumor development is not clear. METHODS AND RESULTS: The expression of ISL-1 was assessed in 211 human lymphoma samples and 23 normal lymph node samples. Immunohistochemistry results demonstrated a markedly higher expression of ISL-1 in 75% of non-Hodgkin lymphoma (NHL) samples compared with that in normal lymph nodes or Hodgkin lymphoma (HL) samples. CCK-8 analysis, cell cycle assay and xenograft model were performed to characterize the association between ISL-1 expression level and biological functions in NHL. The results showed that ISL-1 overexpression obviously promoted NHL cells proliferation, changed the cell cycle distribution in vitro and significantly enhanced xenografted lymphoma development in vivo. Real-time PCR, Western blot, luciferase assay and ChIP assay were used to explore the potential regulatory targets of ISL-1 and the results demonstrated that ISL-1 activated the c-Myc expression in NHL by direct binding to a conserved binding site on the c-Myc enhancer. Further results revealed that ISL-1 could be positively regulated by the c-Jun N-terminal kinase (JNK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. Both the JNK and JAK/STAT signaling inhibitors could significantly suppressed the growth of NHL cells through the down-regulation of ISL-1 as demonstrated by CCK-8 and Western blot assays. Bioinformatic analysis and luciferase assay exhibited that ISL-1 was a novel target of p-STAT3 and p-c-jun. ChIP, Co-IP and ChIP-re-IP analysis revealed that ISL-1 could participate with p-STAT3 and p-c-Jun to form a p-STAT3/p-c-Jun/ISL-1 transcriptional complex that binds directly on the ISL-1 promoter, demonstrating a positive feedback regulatory mechanism for ISL-1 expression in NHL. CONCLUSIONS: Our results provide the first evidence that ISL-1 is tightly linked to NHL proliferation and development by promoting c-Myc transcription, and its aberrant expression was regulated by p-STAT3/p-c-Jun/ISL-1 complex activation.

A positive feedback regulation of ISL-1 in DLBCL but not in pancreatic ß-cells.

Insulin enhancer binding protein-1 (ISL-1), a LIM-homeodomain transcription factor, has been reported to play essential roles in promoting adult pancreatic ß-cells proliferation. Recent studies indicate that ISL-1 may also involve in the occurrence of a variety of tumors. However, whether ISL-1 has any functional effect on tumorigenesis, and what are the differences on ISL-1 function in distinct conditions, are completely unknown. In this study, we found that ISL-1 was highly expressed in human pancreatic ß-cells, as well as in diffuse large B cell lymphoma (DLBCL), but to a much less extent in other normal tissues or tumor specimens. Further study revealed that ISL-1 promoted the proliferation of pancreatic ß-cells and DLBCL cells, and also accelerated the tumorigenesis of DLBCL in vivo. We also found that ISL-1 could activate c-Myc transcription not only in pancreatic ß-cells but also in DLBCL cells. However, a cell-specific feedback regulation was detectable only in DLBCL cells. This auto-regulatory loop was established by the interaction of ISL-1 and c-Myc to form an ISL-1/c-Myc transcriptional complex, and synergistically to promote ISL-1 transcription through binding on the ISL-1 promoter. Taken together, our results demonstrate a positive feedback regulation of ISL-1 in DLBCL but not in pancreatic ß-cells, which might result in the functional diversities of ISL-1 in different physiological and pathological processes.

Wnt-promoted Isl1 expression through a novel TCF/LEF1 binding site and H3K9 acetylation in early stages of cardiomyocyte differentiation of P19CL6 cells.

Islet 1 (ISL1), a marker of second heart field progenitors, plays a crucial role in cardiomyocyte differentiation and proliferation. However, little is known about transcriptional regulating mechanisms on Isl1 gene expression. Recent studies have demonstrated that Wnt/ß-catenin signaling regulates Isl1 expression during heart development. However, the detailed mechanisms still remain unclear. In the present study performed during differentiation of P19CL6 into cardiomyocytes, we explored the underlying regulating mechanisms on Wnt/ß-catenin-mediated Isl1 expression after we first confirmed that Wnt/ß-catenin signaling promoted cardiomyocyte differentiation partly through Isl1 activation. We found a novel TCF/LEF1 binding site that was located 2300 bp upstream of the Isl1 ATG. Furthermore, Wnt/ß-catenin signaling upregulated histone H3K9 acetylation on TCF/LEF1 binding sites on the Isl1 promoter, resulting in upregulation of Isl1 expression. This Wnt-mediated H3K9 acetylation on the Isl1 promoter was modulated by the acetyltransferase CREB-binding protein (CBP), instead of p300, through interaction with ß-catenin. Collectively, these results suggest that in early stages of cardiomyocyte differentiation Wnt/ß-catenin signaling promotes Isl1 expression via two ways: a novel TCF/LEF1 binding site and H3K9 acetylation conducted by CBP on the Isl1 promoter. To our knowledge, this is the first study reporting Wnt/ß-catenin-regulated H3K9 acetylation on promoters of its target genes. And this study gives new insights into transcriptional regulating mechanisms of Wnt-mediated Isl1 expression during cardiomyocyte differentiation.

miR-499 protects cardiomyocytes from H 2O 2-induced apoptosis via its effects on Pdcd4 and Pacs2.

Background microRNAs (miRNAs) are a class of small, non-coding endogenous RNAs that post-transcriptionally regulate some protein-coding genes. miRNAs play an important role in many cardiac pathophysiological processes, including myocardial infarction, cardiac hypertrophy, and heart failure. miR-499, specifically expressed in skeletal muscle and cardiac cells, is differentially regulated and functions in heart development. However, the function of miR-499 in mature heart is poorly understood. Results We report that cardiac-abundant miR-499 could protect neonatal rat cardiomyocytes against H 2O 2-induced apoptosis. Increased miR-499 level favored survival, while decreased miR-499 level favored apoptosis. We identified three proapoptotic protein-coding genes-Pdcd4, Pacs2, and Dyrk2-as targets of miR-499. miR-499 inhibited cardiomyocyte apoptosis through its suppressive effect on Pdcd4 and Pacs2 expression, thereby blocking Bid expression and BID mitochondrial translocation. We also found that H 2O 2-induced phosphorylation of c-Jun transcriptionally upregulated miR-499 expression via binding of phosphorylated c-Jun to the Myh7b promoter. Conclusions Our results revealed that miR-499 played an inhibiting role in the mitochondrial apoptosis pathway, and had protective effects against H 2O 2-induced injury in cardiomyocytes.

Involvement of ERK-RSK cascade in phenylephrine-induced phosphorylation of GATA4.

GATA4 has been characterized as a crucial regulator of cardiac development and hypertrophy. Multiple signaling pathways involving MAPK contribute to GATA4 activation via direct phosphorylation. MSK and RSK are two kinase families mediating signal transduction downstream of the MAPK cascade. In this study, we investigated the effects of MSK and RSK on GATA4 activation. Overexpression of RSK2 greatly increased phosphorylation of GATA4 at Ser261. This phosphorylation enhanced its transcriptional and DNA binding activity. RSK-dependent phosphorylation of GATA4 also led to enhanced interaction with NKX2.5 and p300. Sequential phosphorylation of the ERK-RSK-GATA4 cascade and nuclear accumulation of RSK in cardiomyocytes were observed after phenylephrine treatment. Inhibition of RSK using the small molecule SL0101 abrogated GATA4 phosphorylation at Ser261, ultimately leading to a repression of fetal cardiac genes. Adenovirus-mediated overexpression of MSK1 had no direct effect on GATA4 phosphorylation but increased GATA4 expression. Together with GATA4 phosphorylation at Ser105 by ERK1/2, our findings show dual phosphorylation of GATA4 by the ERK-RSK cascade and suggest that MSK and RSK have distinct effects in PE-induced cardiac hypertrophic response.

Interaction of Wnt/ß-catenin and notch signaling in the early stage of cardiac differentiation of P19CL6 cells.

Notch and Wnt/ß-catenin signaling both play essential roles and interact closely in cardiomyocyte differentiation but the mechanism of interaction is largely unknown. Here we show that activation of Notch signaling in undifferentiated P19CL6 cells promoted cardiac differentiation, indicated by upregulated expression of early cardiac markers and activated the canonical Wnt pathway, suggested by augmented nuclear translocation of ß-catenin. Further activation of the Notch pathway in early differentiating cells (at day 3) inhibited expression of a specific cardiac progenitor marker Islet1 but had no influence on ß-catenin translocation. Notch signaling thus played biphasic roles in the early stage of cardiomyocyte differentiation and Wnt/ß-catenin signaling. Unlike Notch signaling, Wnt signaling promoted cardiomyocyte differentiation and activated the Notch pathway in either undifferentiated or early differentiating cells. Additionally, ß-catenin, recombination signal sequence binding protein-Jkappa (RBP-Jκ), and Notch1 intracellular domain (NICD-1) formed a transcriptional complex which was recruited to the Hes1 promoter region, indicating direct transcriptional regulation of Hes1. We thus document a specific reciprocal interaction between these two signaling pathways during early stage cardiac differentiation of P19CL6 cells.

α-lipoic acid inhibits high glucose-induced apoptosis in HIT-T15 cells.

High blood glucose plays an important role in the pathogenesis of diabetes. α-lipoic acid (LA) has been used to prevent and treat diabetes, and is thought to act by increasing insulin sensitivity in many tissues. However, whether LA also has a cytoprotective effect on pancreatic islet beta cells remains unclear. In this study, we assessed whether LA could inhibit apoptosis in beta cells exposed to high glucose concentrations. HIT-T15 pancreatic beta cells were treated with 30 mmol/L glucose in the presence or absence of 0.5 mmol/L LA for 8 days. LA significantly reduced the numbers of apoptotic HIT-T15 cells and inhibited the cell overgrowth normally induced by high glucose treatment. Additionally, LA inhibited insulin expression and secretion in HIT-T15 cells induced by high glucose. Further study demonstrated that LA upregulated Pdx1 and Bcl2 gene expression, reduced Bax gene expression, and promoted phosphorylation of Akt in HIT-T15 cells treated with high glucose. Intriguingly, knockdown of Pdx1 expression partially offset the anti-apoptotic effect of LA. However, inhibition of Akt by PI3K/AKT antagonist LY294002 only slightly reversed the anti-apoptosis effect of LA and mildly decreased the gene expression level of Pdx1 (P > 0.05). Moreover, LA only slightly attenuated reactive oxygen species (ROS) production and augmented mitochondrial membrane potential. Therefore, our data suggest that α-lipoic acid can effectively attenuate high glucose-induced HIT-T15 cell apoptosis probably by increasing Pdx1 expression. These findings provide a new interpretation on the role of LA in the treatment of diabetes.

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.

GATA4 regulates ANF expression synergistically with Sp1 in a cardiac hypertrophy model.

Cardiac hypertrophy in response to multiple stimuli has important physiological and pathological significances. GATA4 serves as a nuclear integrator of several signalling pathways during cardiac hypertrophy. Sp1 and Sp3 are also reported to be involved in this process. However, the mechanism by which GATA4 acts as a mediator, integrating these ubiquitously expressed transcriptional factors, is poorly understood. We found that the expression of GATA4 and Sp1 was up-regulated in the myocardium of a pressure overload hypertrophy rat model, as well in phenylephrine-induced (PE-induced) hypertrophic growth of neonatal cardiomyocytes. GST pull-down assays demonstrated that GATA4 could interact with Sp1 in vitro. Therefore, we proposed that GATA4 cooperates with Sp1 in regulating ANF expression, as its reactivation is closely linked with hypertrophy. Further studies demonstrated that GATA4 could activate the ANF promoter synergistically with Sp1 through direct interaction. In contrast, Sp3 exhibited antagonistic function, and overexpression of Sp3 repressed the transcriptional synergy between Sp1 and GATA4. We also found that Sp1 alone could activate the ANF promoter in cardiomyocytes, whereas Sp3 exerted negative effects on ANF expression. Bioinformatics analysis revealed novel Sp-binding sites on the ANF promoter. The recruitment of GATA4 and Sp1 on the ANF promoter was enhanced during phenylephrine-mediated hypertrophy, whereas the recruitment of Sp3 was reduced. The phosphorylation of GATA4 by ERK1/2 kinase could enhance the affinity between GATA4 and Sp1. Thus, our findings revealed the critical interaction of GATA4 and Sp1 in modulating ANF expression, indicating their involvement in cardiac hypertrophy.

Inhibitor of DNA binding 1 (Id1) induces differentiation and proliferation of mouse embryonic carcinoma P19CL6 cells.

The inhibitor of DNA binding (Id) family of genes encodes negative regulators of basic helix-loop-helix transcription factors and has been implicated in such diverse cellular processes as differentiation, proliferation, apoptosis and migration. Id knockout mouse embryos display multiple cardiac defects but the specific role of Id1 in cardiac differentiation is unclear. In the present study, we investigated the function of Id1 in DMSO-induced P19CL6 cells, a widely-accepted cell model of cardiac differentiation. We found that Id1 was upregulated during the cardiac differentiation of P19CL6 cells. The expression of cardiac specific marker genes, Gata4, α-MHC and ISL1, was upregulated in P19CL6 cells stably transfected with Id1 (P19CL6-Id1) during cardiac differentiation. The overexpression of Id1 reduced the number of cells in G1 phase and increased the cell population in G2, M and S phases, while knockdown of Id1 increased the number of cells in G1 phase from 48.6 ± 2.51% to 62.2 ± 1.52% at day 0 of cardiac induction, and from 52.5 ± 3.41% to 63.7 ± 1.02% at day 3 after cardiac induction, indicating that Id1 promoted proliferation of P19CL6 cells. Luciferase assays showed that the activity of TOP flash was higher in P19CL6-Id1 cells than wildtype P19CL6 cells, while Id1 expression was also upregulated in P19CL6 cells treated with Wnt3a or LiCl. This indicates that there may be positive feedback between Id1 and Wnt signaling which plays an important role in cardiac differentiation.

Requirement of the NF-κB pathway for induction of Wnt-5A by interleukin-1ß in condylar chondrocytes of the temporomandibular joint: functional crosstalk between the Wnt-5A and NF-κB signaling pathways.

OBJECTIVE: We have previously reported that interleukin-1ß (IL-1ß) up-regulates the expression of Wnt-5A and the activation of Wnt-5A signaling induces matrix metalloproteinase (MMP) through the c-Jun N-terminal kinase pathway in condylar chondrocytes (CCs) of the temporomandibular joint (TMJ). These results suggest that Wnt-5A could play an essential role in IL-1ß-mediated cartilage destruction. The objective of this study was to investigate the molecular mechanism underlying IL-1ß-induced up-regulation of Wnt-5A in TMJ CCs. METHODS: Primary CCs, limb chondrocytes (LCs) and SW1353 human chondrosarcoma cells were treated with IL-1ß in the presence or absent of BAY 11-7082 (an inhibitor of IκBα-phosphorylation). Then, expression of Wnt-5A was estimated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunocytofluorescence. Transient transfection of p65 expression vector and chromatin immunoprecipitation (ChIP) assay was performed to define the effect of p65 on Wnt-5A expression. RESULTS: IL-1ß up-regulated Wnt-5A expression at both the RNA and protein levels in articular chondrocytes. The inhibitor of IκBα-phosphorylation, BAY 11-7082, blocked the induction of Wnt-5A by IL-1ß in a dose-dependent manner. Moreover, experiments with overexpression of p65 and ChIP established that induction of Wnt-5A by IL-1ß is mediated through the NF-κB pathway, especially the p65 subunit. CONCLUSION: These results clarify the molecular mechanism underlying up-regulation of Wnt-5A by IL-1ß in chondrocytes, suggesting an important functional crosstalk between Wnt-5A and NF-κB signaling pathways. This finding provides new insights into the involvement of Wnt signaling in the cartilage destruction caused by arthritis.

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.

Beta-catenin/LEF1 activated enamelin expression in ameloblast-like cells.

Enamelin is an ameloblast-specific matrix protein believed to play essential roles in enamel formation. However, mechanisms of enamelin transcription regulation are not clear. beta-Catenin/LEF1 is a key transcriptional complex involved in tooth development. In this study, the role of beta-catenin/LEF1 in enamelin expression was investigated. The 5'-flanking region of the mouse enamelin gene was analyzed and cloned. Co-transfection analysis and mutation assays revealed that two conserved LEF1 responsive elements located at -1002 and -597bp upstream of the enamelin translation initiation site could augment transcriptional activity of the enamelin. The interaction between the enamelin elements and beta-catenin/LEF1 was further confirmed by electrophoresis mobility shift assays and chromatin immunoprecipitation assays. In addition, LiCl treatment induced nuclear translocation of beta-catenin and elevated endogenous enamelin expression in mouse ameloblast-like cells. The results suggested that Wnt/beta-catenin signaling could function in enamelin gene expression by direct interaction through two conserved LEF1 responsive elements on the enamelin gene in ameloblast-like cells.

Angiopoietin-1/Tie2 signaling pathway inhibits lipopolysaccharide-induced activation of RAW264.7 macrophage cells.

Angiopoietin-1 (Ang1) is a ligand for the endothelial-specific tyrosine kinase receptor Tie2 and has been shown to play an essential role in embryonic vasculature development. There have been many studies about the anti-inflammatory effects of Ang1, most of which focus on endothelium cells. In the present study, we explore the role of Ang1-Tie2 signaling in the activation of macrophages upon lipopolysaccharide (LPS) stimulation. We found that Tie2 receptor is expressed on macrophages and Ang1 could inhibit LPS-induced activation of macrophages, as evidenced by cell migration and TNF-alpha production, specifically through Tie2 receptor. We further investigated the mechanism and found that Ang1-Tie2 could block LPS-induced activation of NF-kappaB which has been shown to be necessary for macrophage activation with LPS treatment. Thus, we described, for the first time, the role of Ang1-Tie2 signaling in macrophage activation and the possible mechanisms in response to immune stimulation.

beta-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.

[Redox factor 1 increases proliferation of neonatal rat cardiac fibroblasts].

OBJECTIVE: To investigate the function of REF1 in the proliferation and collagen synthesis of neonatal rat cardiac fibroblasts, and the underlying mechanisms. METHODS: Neonatal rat cardiac fibroblasts were transfected with the adenoviral vector containing rat wild type Ref1 (Ad-Ref1) or mutated Ref1 (Ad-mutRef1). The mutations resulted in Cys to Ala at amino acids 65 and 93, which eliminated the redox function of the REF1 protein. MTT was used to check the cell viability and flow cytometry was used to analyze the cell proliferation with the count of cell numbers and the percentage of cells in S phase of the cell cycle. The expressions of Ref1, collagen I (Col I) and collagen III (Col III) were determined by RT-PCR and Western blot. The translocation of REF1 was examined by fluorescence staining and revealed under fluorescence microscope. Electrophoretic mobility shift assay (EMSA) was used to check the effect of REF1 on AP1 DNA binding ability. The high glucose medium (25 mmol/L) was applied to culture cardiac fibroblasts. The effect of high glucose on AP1 DNA binding activity, the expression and translocation of REF1 were examined. RESULTS: MTT analysis showed that Ad-Ref1 promoted the relative viability of cardiac fibroblasts (0.671+/-0.044 vs control 0.364+/-0.007, n=6, P<0.01). The percentage of cells in S phase of the cell cycle was increased significantly in the Ad-Ref1 transfected cells (16.8%+/-0.62% vs control 9.04%+/-0.43%, n=3, P<0.05), as demonstrated by flow cytometry analysis. The expressions of Col I and Col III at mRNA level were increased when cells transfected with Ad-Ref1, while Ad-mutRef1 did not show such effects. Compared with the redox-deficient mutant Ad-mutRef1 (C65/93A), EMSA results demonstrated that Ad-Ref1 resulted in a marked increase in AP1 DNA binding. We also found that the cardiac fibroblasts cultured in high glucose (25 mmol/L) medium resulted in an increase in AP1 DNA binding activity, which was similar as seen in Ad-Ref1 transfected cells. There was also an increased accumulation of nuclear REF1 protein when cells were cultured in high glucose medium, although the expressions of REF1 at both mRNA and protein levels were not affected. CONCLUSION: REF1 can increase proliferation and collagen synthesis of cardiac fibroblasts, which may be related to its ability to up-regulate AP1 DNA binding.

Mouse embryonic stem cell-derived cardiomyocytes express functional adrenoceptors.

The cardiogenic capacity of embryonic stem (ES) cells has been well-investigated. However, little is known about the development of adrenoceptor (AR) systems during the process of ES cell differentiation, which are critically important in cardiac physiology and pharmacology. In this present study, we investigated the expression profile of adrenoceptor subtypes, beta-adrenergic modulation of muscarinic receptors and adrenoceptor-related signaling in cardiomyocytes derived from ES cells (ESCMs). Reverse transcription-polymerase chain reaction revealed that undifferentiated mouse ES cells expressed alpha(1A)-, alpha(1B)-, alpha(1D)- and beta(2)-AR mRNA. However, beta(1)-AR was only expressed after vitamin C induction. The expressions of alpha(1A)-, alpha(1D)- and beta(1)-ARs increased significantly while alpha(1B)- and beta(2)-ARs showed no significant change during the differentiation process. Furthermore, we detected the expression of tyrosine hydroxylase. Both alpha(1)-AR and beta-AR could activate extracellular responsive kinase in ESCMs. Isoprenaline could inhibit the expression of M(2) muscarinic receptor protein. CGP20712A, a beta(1)-AR antagonist, up-regulated the expression of M(2) muscarinic receptor while ICI118551, a beta(2)-AR antagonist, showed no effect. These results indicated that functional adrenoceptors and tyrosine hydroxylase, a critical enzyme in catecholamine biosynthesis, were differentially expressed in ESCMs. Adrenoceptor-related signaling pathways and beta-adrenergic modulation of muscarinic receptors were established during differentiation.

Carboxyl terminus of Nkx2.5 impairs its interaction with p300.

The transcription factor Nkx2.5 plays critical roles in controlling cardiac-specific gene expression. Previous reports demonstrated that Nkx2.5 is only a modest transactivator due to the auto-inhibitory effect of its C-terminal domain. Deletion of the C-terminal domain, mimicking conformational change, evokes vigorous transactivation activity. Here, we show that a C-terminal defective mutant of Nkx2.5 improves the occupation of p300 at the ANF promoter compared with full-length Nkx2.5, leading to hyperacetylation of histone H4. We reveal that p300 is a cofactor of Nkx2.5, markedly potentiating Nkx2.5-dependent transactivation, whereas E1A antigen impairs Nkx2.5 activity. Furthermore, p300 can acetylate Nkx2.5 and display an acetyltransferase-independent mechanism to coactivate Nkx2.5. Physical interaction between the N-terminal activation domain of Nkx2.5 and the C/H3 domain of p300 are identified by GST pull-down assay. Point mutants of the N-terminal modify the transcriptional activity of Nkx2.5 and interaction with p300. Deletion of the C-terminal domain greatly facilitates p300 binding and improves the susceptibility of Nkx2.5 to histone deacetylase inhibitor. These results establish that p300 acts as an Nkx2.5 cofactor and facilitates increased Nkx2.5 activity by relieving the conformational impediment of its inhibitory C-terminal domain.