Label-Free Assessment of Neuronal Activity Using Raman Micro-Spectroscopy.

Given the pivotal role of neuronal populations in various biological processes, assessing their collective output is crucial for understanding the nervous system's complex functions. Building on our prior development of a spiral scanning mechanism for the rapid acquisition of Raman spectra from single cells and incorporating machine learning for label-free evaluation of cell states, we investigated whether the Paint Raman Express Spectroscopy System (PRESS) can assess neuronal activities. We tested this hypothesis by examining the chemical responses of glutamatergic neurons as individual neurons and autonomic neuron ganglia as neuronal populations derived from human-induced pluripotent stem cells. The PRESS successfully acquired Raman spectra from both individual neurons and ganglia within a few seconds, achieving a signal-to-noise ratio sufficient for detailed analysis. To evaluate the ligand responsiveness of the induced neurons and ganglia, the Raman spectra were subjected to principal component and partial least squares discriminant analyses. The PRESS detected neuronal activity in response to glutamate and nicotine, which were absent in the absence of calcium. Additionally, the PRESS induced dose-dependent neuronal activity changes. These findings underscore the capability of the PRESS to assess individual neuronal activity and elucidate neuronal population dynamics and pharmacological responses, heralding new opportunities for drug discovery and regenerative medicine advancement.

Isolation of TCR genes with tumor-killing activity from tumor-infiltrating and circulating lymphocytes in a tumor rejection cynomolgus macaque model.

To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and ß pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models.

ETS-dependent enhancers for endothelial-specific expression of serum/glucocorticoid-regulated kinase 1 during mouse embryo development.

Serum/glucocorticoid-regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5' region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500 kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS-regulated enhancers located in the proximal and distal regions.

A role of Hey2 transcription factor for right ventricle development through regulation of Tbx2-Mycn pathway during cardiac morphogenesis.

A basic helix-loop-helix transcription factor Hey2 is expressed in the ventricular myocardium and endocardium of mouse embryos, and Hey2 null mice die perinatally showing ventricular septal defect, dysplastic tricuspid valve and hypoplastic right ventricle. In order to understand region-specific roles of Hey2 during cardiac morphogenesis, we generated Hey2 conditional knockout (cKO) mice using Mef2c-AHF-Cre, which was active in the anterior part of the second heart field and the right ventricle and outflow tract of the heart. Hey2 cKO neonates reproduced three anomalies commonly observed in Hey2 null mice. An earliest morphological defect was the lack of right ventricular extension along the apico-basal axis at midgestational stages. Underdevelopment of the right ventricle was present in all cKO neonates including those without apparent atresia of right-sided atrioventricular connection. RNA sequencing analysis of cKO embryos identified that the gene expression of a non-chamber T-box factor Tbx2 was ectopically induced in the chamber myocardium of the right ventricle. Consistently, mRNA expression of the Mycn transcription factor, which was a cell cycle regulator transcriptionally repressed by Tbx2, was down regulated, and the number of S-phase cells was significantly decreased in the right ventricle of cKO heart. These results suggest that Hey2 plays an important role in right ventricle development during cardiac morphogenesis, at least in part, through mitigating Tbx2-dependent inhibition of Mycn expression.

Negatively charged amino acids in the stalk region of membrane proteins reduce ectodomain shedding.

Ectodomain shedding is a post-translational modification mechanism by which the entire extracellular domain of membrane proteins is liberated through juxtamembrane processing. Because shedding rapidly and irreversibly alters the characteristics of cells, this process is properly regulated. However, the molecular mechanisms governing the propensity of membrane proteins to shedding are largely unknown. Here, we present evidence that negatively charged amino acids within the stalk region, an unstructured juxtamembrane region at which shedding occurs, contribute to shedding susceptibility. We show that two activated leukocyte cell adhesion molecule (ALCAM) protein variants produced by alternative splicing have different susceptibilities to ADAM metallopeptidase domain 17 (ADAM17)-mediated shedding. Of note, the inclusion of a stalk region encoded by a 39-bp-long alternative exon conferred shedding resistance. We found that this alternative exon encodes a large proportion of negatively charged amino acids, which we demonstrate are indispensable for conferring the shedding resistance. We also show that the introduction of negatively charged amino acids into the stalk region of shedding-susceptible ALCAM variant protein attenuates its shedding. Furthermore, we observed that negatively charged amino acids residing in the stalk region of Erb-B2 receptor tyrosine kinase 4 (ERBB4) are indispensable for its shedding resistance. Collectively, our results indicate that negatively charged amino acids within the stalk region interfere with the shedding of multiple membrane proteins. We conclude that the composition of the stalk region determines the shedding susceptibility of membrane proteins.

Metabolic remodeling during somatic cell reprogramming to induced pluripotent stem cells: involvement of hypoxia-inducible factor 1.

Induced pluripotent stem cells (iPSCs) were first established from differentiated somatic cells by gene introduction of key transcription factors, OCT4, SOX2, KLF4, and c-MYC, over a decade ago. Although iPSCs can be applicable for regenerative medicine, disease modeling and drug screening, several issues associated with the utilization of iPSCs such as low reprogramming efficiency and the risk of tumorigenesis, still need to be resolved. In addition, the molecular mechanisms involved in the somatic cell reprogramming to pluripotency are yet to be elucidated. Compared with their somatic counterparts, pluripotent stem cells, including embryonic stem cells and iPSCs, exhibit a high rate of glycolysis akin to aerobic glycolysis in cancer cells. This is known as the Warburg effect and is essential for maintaining stem cell properties. This unique glycolytic metabolism in iPSCs can provide energy and drive the pentose phosphate pathway, which is the preferred pathway for rapid cell proliferation. During reprogramming, somatic cells undergo a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis trigged by a transient OXPHOS burst, resulting in the initiation and progression of reprogramming to iPSCs. Metabolic intermediates and mitochondrial functions are also involved in the epigenetic modification necessary for the process of iPSC reprogramming. Among the key regulatory molecules that have been reported to be involved in metabolic shift so far, hypoxia-inducible factor 1 (HIF1) controls the transcription of many target genes to initiate metabolic changes in the early stage and maintains glycolytic metabolism in the later phase of reprogramming. This review summarizes the current understanding of the unique metabolism of pluripotent stem cells and the metabolic shift during reprogramming, and details the relevance of HIF1 in the metabolic shift.

Serum/glucocorticoid-regulated kinase 1 as a novel transcriptional target of bone morphogenetic protein-ALK1 receptor signaling in vascular endothelial cells.

Bone morphogenetic protein 9 (BMP9)/BMP10-ALK1 receptor signaling is essential for endothelial differentiation and vascular morphogenesis. Mutations in ALK1/ACVRL1 and other signal-related genes are implicated in human vascular diseases, and the Alk1/Acvrl1 deletion in mice causes severe impairment of vascular formation and embryonic lethality. In the microarray screen to search for novel downstream genes of ALK1 signaling, we found that the mRNA and protein expression of serum/glucocorticoid-regulated kinase 1 (SGK1) was rapidly up-regulated by the BMP9 stimulation of cultured human endothelial cells. The increase in SGK1 mRNA was completely blocked by the transcriptional inhibitor actinomycin D and significantly suppressed by the siRNA treatment against the co-SMAD transcription factor SMAD4. Upon the BMP9 treatment of endothelial cells, phosphorylated SMAD1/5/9 bound to a consensus site upstream of the SGK1 gene, which was necessary for BMP9-dependent increment of the luciferase reporter activity driven by the SGK1 proximal enhancer. The Sgk1 mRNA expression in mouse embryos was enriched in vascular endothelial cells at embryonic day 9.0-9.5, at which Sgk1 null mice showed embryonic lethality due to abnormal vascular formation, and its mRNA as well as protein expression was clearly reduced in Alk1/Acvrl1 null embryos. These results indicate that SGK1 is a novel target gene of BMP9/BMP10-ALK1 signaling in endothelial cells and further suggest a possibility that down-regulation of the Sgk1 expression may be involved in the mechanisms of vascular defects by the ALK1 signaling deficiency.

ERRs Mediate a Metabolic Switch Required for Somatic Cell Reprogramming to Pluripotency.

Cell metabolism is adaptive to extrinsic demands; however, the intrinsic metabolic demands that drive the induced pluripotent stem cell (iPSC) program remain unclear. Although glycolysis increases throughout the reprogramming process, we show that the estrogen-related nuclear receptors (ERRα and ERRγ) and their partnered co-factors PGC-1α and PGC-1ß are transiently induced at an early stage, resulting in a burst of oxidative phosphorylation (OXPHOS) activity. Upregulation of ERRα or ERRγ is required for the OXPHOS burst in both human and mouse cells, respectively, as well as iPSC generation itself. Failure to induce this metabolic switch collapses the reprogramming process. Furthermore, we identify a rare pool of Sca1(-)/CD34(-) sortable cells that is highly enriched in bona fide reprogramming progenitors. Transcriptional profiling confirmed that these progenitors are ERRγ and PGC-1ß positive and have undergone extensive metabolic reprogramming. These studies characterize a previously unrecognized, ERR-dependent metabolic gate prior to establishment of induced pluripotency.

Foxd1 is a mediator and indicator of the cell reprogramming process.

It remains unclear how changes in gene expression profiles that establish a pluripotent state are induced during cell reprogramming. Here we identify two forkhead box transcription factors, Foxd1 and Foxo1, as mediators of gene expression programme changes during reprogramming. Knockdown of Foxd1 or Foxo1 reduces the number of iPSCs, and the double knockdown further reduces it. Knockout of Foxd1 inhibits downstream transcriptional events, including the expression of Dax1, a component of the autoregulatory network for maintaining pluripotency. Interestingly, the expression level of Foxd1 is transiently increased in a small population of cells in the middle stage of reprogramming. The transient Foxd1 upregulation in this stage is correlated with a future cell fate as iPSCs. Fate mapping analyses further reveal that >95% of iPSC colonies are derived from the Foxd1-positive cells. Thus, Foxd1 is a mediator and indicator of successful progression of reprogramming.

Cell line-dependent differentiation of induced pluripotent stem cells into cardiomyocytes in mice.

AIMS: Mouse and human fibroblasts can be directly reprogrammed to pluripotency by the ectopic expression of four transcription factors (Oct3/4, Sox2, Klf4, and c-Myc) to yield induced pluripotent stem (iPS) cells. iPS cells can be generated even without the expression of c-Myc. The present study examined patterns of differentiation of mouse iPS cells into cardiomyocytes in three different cell lines reprogrammed by three or four factors. METHODS AND RESULTS: During the induction of differentiation on feeder-free gelatinized dishes, genes involved in cardiogenesis were expressed as in embryonic stem cells and myogenic contraction occurred in two iPS cell lines. However, in one iPS cell line (20D17) generated by four factors, the expression of cardiac-specific genes and the beating activity were extremely low. Treating iPS cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased Nkx2.5 expression in all iPS cell lines. While the basal Nkx2.5 expression was very low in 20D17, the TSA-induced increase was the greatest. TSA also induced the expression of contractile proteins in 20D17. Furthermore, we demonstrated the increased mRNA level of Oct3/4 and nuclear protein level of HDAC4 in 20D17 compared with the other two iPS cell lines. DNA microarray analysis identified genes whose expression is up- or down-regulated in 20D17. CONCLUSIONS: Mouse iPS cells differentiate into cardiomyocytes in a cell line-dependent manner. TSA induces myocardial differentiation in mouse iPS cells and might be useful to overcome cell line variation in the differentiation efficiency.

Soluble VEGF receptor-2 is increased in sera of subjects with metabolic syndrome in association with insulin resistance.

OBJECTIVE: Metabolic syndrome (MetS) is associated with impaired angiogenesis. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis through binding to its specific receptor, VEGF receptor-2 (VEGFR-2), whereas the expression of VEGF and VEGFR-2 in the myocardium of insulin-resistant rats is down-regulated. Soluble VEGF receptor-1 (sVEGFR-1) and -2 (sVEGFR-2) have been reported to inhibit angiogenesis both in vitro and in vivo. However, the balance between circulating levels of VEGF and its soluble receptors, which may reflect and/or affect cardiovascular VEGF signaling, in subjects with MetS is unknown. METHODS AND RESULTS: We carried out a cross-sectional study including 272 consecutive, apparently healthy subjects who were not receiving any drugs. Plasma levels of VEGF and serum levels of its soluble receptors were determined using enzyme-linked immunosorbent assays. VEGF and sVEGFR-1 levels did not differ between subjects with and those without MetS. However, sVEGFR-2 levels were significantly increased in MetS compared with non-MetS subjects. Stepwise regression analysis revealed that HOMA-IR was the strongest independent determinant of the sVEGFR-2 level. Accordingly, the mean sVEGFR-2 levels increased in proportion to both the accumulation of components of MetS and quartile of HOMA-IR. Interestingly, multiple regression analyses revealed that independent determinants of VEGF were the body mass index and blood pressure, whereas, in contrast, those of sVEGFR-2 were HOMA-IR and high-sensitivity C-reactive protein. CONCLUSIONS: The correlation of sVEGFR-2 with insulin resistance supports the need for further investigations to define the clinical utility and predictive value of serum sVEGFR-2 levels in cardiovascular dysfunction in MetS.

Linking the p53 tumour suppressor pathway to somatic cell reprogramming.

Reprogramming somatic cells to induced pluripotent stem (iPS) cells has been accomplished by expressing pluripotency factors and oncogenes, but the low frequency and tendency to induce malignant transformation compromise the clinical utility of this powerful approach. We address both issues by investigating the mechanisms limiting reprogramming efficiency in somatic cells. Here we show that reprogramming factors can activate the p53 (also known as Trp53 in mice, TP53 in humans) pathway. Reducing signalling to p53 by expressing a mutated version of one of its negative regulators, by deleting or knocking down p53 or its target gene, p21 (also known as Cdkn1a), or by antagonizing reprogramming-induced apoptosis in mouse fibroblasts increases reprogramming efficiency. Notably, decreasing p53 protein levels enabled fibroblasts to give rise to iPS cells capable of generating germline-transmitting chimaeric mice using only Oct4 (also known as Pou5f1) and Sox2. Furthermore, silencing of p53 significantly increased the reprogramming efficiency of human somatic cells. These results provide insights into reprogramming mechanisms and suggest new routes to more efficient reprogramming while minimizing the use of oncogenes.

Myocardial regulation of p300 and p53 by doxorubicin involves ubiquitin pathways.

BACKGROUND: Doxorubicin (Dox) depletes p300 from cardiac myocytes and induces apoptosis of these cells. p300 protein possesses ubiquitin ligase activity for the p53 tumor suppressor gene product, catalyzes p53 polyubiqutination, and facilitates p53 degradation in an ubiquitin-dependent manner. The present study investigated the ubiquitin-dependent regulation of p53 by Dox and p300 in cardiac myocytes. METHODS AND RESULTS: Primary cardiac myocytes from neonatal rats were exposed to a proteasome inhibitor, MG132, in culture. MG132 increased both p300 and p53 protein levels in these cells, suggesting that ubiquitin-dependent degradation is involved in the homeostasis of these proteins. Notably, treatment of cardiac myocytes with Dox decreased the protein levels of p300 but markedly increased those of p53. By immunoprecipitation-Western blotting, it was shown that treatment with Dox decreased poly-ubiquitinated p53 but increased that of p300 in cardiac myocytes. Finally, the overexpression of p300 in cardiomyocytes suppressed the Dox-mediated increase in the p53 level in addition to inhibiting Dox-induced apoptosis. CONCLUSION: Dox reciprocally regulates p300 and p53 through ubiquitin-dependent pathways and that p300, by its ubiquitin ligase activity, is partially involved in the ubiquitin-dependent degradation of p53 in cardiac myocytes.

The dietary compound curcumin inhibits p300 histone acetyltransferase activity and prevents heart failure in rats.

Hemodynamic overload in the heart can trigger maladaptive hypertrophy of cardiomyocytes. A key signaling event in this process is nuclear acetylation by histone deacetylases and p300, an intrinsic histone acetyltransferase (HAT). It has been previously shown that curcumin, a polyphenol responsible for the yellow color of the spice turmeric, possesses HAT inhibitory activity with specificity for the p300/CREB-binding protein. We found that curcumin inhibited the hypertrophy-induced acetylation and DNA-binding abilities of GATA4, a hypertrophy-responsive transcription factor, in rat cardiomyocytes. Curcumin also disrupted the p300/GATA4 complex and repressed agonist- and p300-induced hypertrophic responses in these cells. Both the acetylated form of GATA4 and the relative levels of the p300/GATA4 complex markedly increased in rat hypertensive hearts in vivo. The effects of curcumin were examined in vivo in 2 different heart failure models: hypertensive heart disease in salt-sensitive Dahl rats and surgically induced myocardial infarction in rats. In both models, curcumin prevented deterioration of systolic function and heart failure-induced increases in both myocardial wall thickness and diameter. From these results, we conclude that inhibition of p300 HAT activity by the nontoxic dietary compound curcumin may provide a novel therapeutic strategy for heart failure in humans.

Oxidative stress induces GLUT4 translocation by activation of PI3-K/Akt and dual AMPK kinase in cardiac myocytes.

In response to metabolic stress, GLUT4, the most abundant glucose transporter, translocates from intracellular vesicles to the plasma membrane. This appears to play an important role in protecting cardiac myocytes from ischemic injury. To investigate the precise mechanisms of GLUT4 translocation in cardiomyocytes, we have established a method for quantifying the relative proportion of sarcolemmal GLUT4 to total GLUT4 in these cells. Stimulation with H2O2 resulted in a concentration-dependent increase in GLUT4 translocation, which peaked at 15 min after stimulation. The dominant-negative form (DN) of AMP-activated protein kinase (AMPK) alpha2 inhibited the H2O2-induced translocation of GLUT4. We further examined the role of two known AMPK kinases (AMPKKs), calmodulin-dependent protein kinase kinase (CaMKK)beta and LKB1. The DN of CaMKKbeta or LKB1 alone inhibited H2O2-induced GLUT4 translocation only partially compared to the inhibition produced by the DN of AMPKalpha2. However, the combination of DN-LKB1 and DN-CaMKKbeta inhibited translocation to an extent similar to with DN-AMPKalpha2. Stimulation with H2O2 also activated Akt and the inhibition of PI3-K/Akt prevented GLUT4 translocation to the same extent as with AMPK inhibition. When the DN of AMPKalpha2 was applied with DN-PI3-K, there was a complete reduction in the GLUT4 membrane level similar to that seen at the 0 time-point. These results demonstrate that AMPK and PI3-K/Akt have an additive effect on oxidative stress-mediated GLUT4 translocation.

Insulin-like growth factor-1 enhances the efficacy of myoblast transplantation with its multiple functions in the chronic myocardial infarction rat model.

BACKGROUND: Myoblast transplantation (Tx) is promising for the improvement of cardiac function in ischemic cardiomyopathy. Insulin-like growth factor-1 (IGF-1) has anti-apoptotic and angiogenic effects, and induces myocyte hypertrophy. Our hypothesis is that topical and slow-release IGF-1 enhances the efficacy of Tx through its multiple functions. METHODS: Four weeks after coronary artery ligation, Lewis rats were divided into four groups: (1) IGF-1+Tx, injection of 6 x 10(6) myoblasts into the infarcted area with placement of an IGF-1-impregnated sheet on the left ventricular (LV) free wall; (2) Tx, Tx alone; (3) IGF-1, IGF-1 sheet alone; and (4) control. We measured cardiac function and performed immunohistochemical examinations. RESULTS: At 4 weeks after treatment, LV diastolic dimension was the smallest, end-systolic elastance was the highest, and tau was the smallest in the IGF-1+Tx group. The graft volume in the IGF-1+Tx group was 3-fold larger than in the Tx group. One day after transplantation, TUNEL-positive donor cells were fewer in the IGF-1+Tx than in the Tx group. Western blot analysis demonstrated that the phosphorylation of Akt increased and the expression of Bax decreased in the transplanted area of IGF-1+Tx rats compared with Tx rats. The vascular density in the peri-infarcted area was larger in IGF-1+Tx than in Tx rats. The mean diameter of graft-derived myotubes was larger in IGF-1+Tx than in Tx animals. CONCLUSIONS: IGF-1 increases the graft volume and enhances the efficacy of Tx in the chronic myocardial infarction model due to its multiple effects of preventing apoptosis, inducing angiogenesis, and promoting myoblast growth.

Activators of PPARgamma antagonize protection of cardiac myocytes by endothelin-1.

Endothelin-1 (ET-1) is a potent survival factor against myocardial cell apoptosis. This anti-apoptotic effect of ET-1 is mediated in part through calcineurin/NFATc-dependent induction of bcl-2 expression. Since it has been reported that peroxisome proliferator-activated receptor-gamma (PPARgamma) interacts with NFATc, we investigated the effects of PPARgamma ligands on anti-apoptotic effects of ET-1 in cardiac myocytes. In primary cardiac myocytes from neonatal rats, administration of PPARgamma activators (15-deoxy-delta12,14-prostaglandin J2 and troglitazone) attenuated the anti-apoptotic effects of ET-1. These activators abolished the ET-1-stimulated increase in bcl-2 expression and in binding of cardiac NFATc to the bcl-2 NFAT site. These findings demonstrate that activators of PPARgamma perturb the anti-apoptotic effects of ET-1 in cardiac myocytes and that this perturbation is, in part, based on functional transcriptional cross-talk between NFATc and PPARgamma.

Endothelin-1-dependent nuclear factor of activated T lymphocyte signaling associates with transcriptional coactivator p300 in the activation of the B cell leukemia-2 promoter in cardiac myocytes.

Endothelin-1 (ET-1) is a potent survival factor that protects cardiac myocytes from apoptosis. ET-1 induces cardiac gene transcription and protein expression of antiapoptotic B cell leukemia-2 (bcl-2) in a calcineurin-dependent manner. A cellular target of adenovirus early region 1A (E1A) oncoprotein, p300 also activates bcl-2 transcription in cardiac myocytes and is required for their survival. p300 acts as a calcineurin-regulated nuclear factors of activated T lymphocytes (NFATc), downstream targets of calcineurin. In addition, the bcl-2 promoter contains multiple NFAT consensus sequences. These findings prompted us to investigate the role of NFATc in ET-1-dependent and p300-dependent bcl-2 transcription in cardiac myocytes. In primary cardiac myocytes prepared from neonatal rats, mutation of 2 NFAT sites within the bcl-2 promoter completely abolished the ET-1- and p300-induced increases in the activity of this promoter. We show here that p300 markedly potentiates the binding of NFATc1 to the bcl-2 NFAT element by interacting with NFATc1 in an E1A-dependent manner. On the other hand, stimulation of cardiac myocytes with ET-1 causes nuclear translocation of NFATc1, which interacts with p300 and increases DNA binding. Expression of E1A did not change the cardiac nuclear localization of NFATc1 but blocked its interaction with p300, DNA binding, and bcl-2 promoter activation. These findings suggest that ET-1-dependent NFATc signaling associates with p300 in the transactivation of bcl-2 gene in cardiac myocytes.

Contribution of caveolin-1 alpha and Akt to TNF-alpha-induced cell death.

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-alpha (TNF-alpha)-resistant lines from L929 cells. Using this approach, we discovered that caveolin-1 alpha is required for TNF-alpha-induced cell death in L929 cells. The need for caveolin-1 alpha in TNF-alpha-induced cell death was confirmed by the restoration of sensitivity to TNF-alpha after ectopic reconstitution of caveolin-1 alpha/beta expression. This caveolin-1 alpha-mutated line was also resistant to H(2)O(2) and staurosporine, but not to lonidamine. HepG2 cells are known to lack endogenous caveolins. HepG2 cells stably transfected with caveolin-1 alpha/beta were found to be much more sensitive to TNF-alpha than either parental cells transfected with caveolin-1 beta or parental cells transfected with an empty vector. In contrast to its extensively documented antiapoptotic effect, the elevated activity of Akt appears to be important in sensitizing caveolin-1-expressing cells to TNF-alpha, since pretreatment of cells with the phosphatidylinositide 3-kinase (PI3K) inhibitor LY-294002 or wortmannin completely blocked PI3K activation and markedly improved the survival of TNF-alpha-treated L929 cells. The survival rates of caveolin-1 alpha-normal and caveolin-1 alpha-deficient L929 cells were comparable after treatment with PI3K inhibitor and TNF-alpha. Similar results were obtained with HepG2 cells that stably expressed caveolin-1 alpha/beta or -beta and parental cells transfected with an empty vector. In summary, our results indicate that caveolin-1 alpha preferentially sensitizes L929 cells to TNF-alpha through the activation of a PI3K/Akt signaling pathway.

Expression of p300 protects cardiac myocytes from apoptosis in vivo.

Doxorubicin is an anti-tumor agent that represses cardiac-specific gene expression and induces myocardial cell apoptosis. Doxorubicin depletes cardiac p300, a transcriptional coactivator that is required for the maintenance of the differentiated phenotype of cardiac myocytes. However, the role of p300 in protection against doxorubicin-induced apoptosis is unknown. Transgenic mice overexpressing p300 in the heart and wild-type mice were subjected to doxorubicin treatment. Compared with wild-type mice, transgenic mice exhibited higher survival rate as well as more preserved left ventricular function and cardiac expression of alpha-sarcomeric actin. Doxorubicin induced myocardial cell apoptosis in wild-type mice but not in transgenic mice. Expression of p300 increased the cardiac level of bcl-2 and mdm-2, but not that of p53 or other members of the bcl-2 family. These findings demonstrate that overexpression of p300 protects cardiac myocytes from doxorubicin-induced apoptosis and reduces the extent of acute heart failure in adult mice in vivo.