GCN5 maintains muscle integrity by acetylating YY1 to promote dystrophin expression.

Protein lysine acetylation is a post-translational modification that regulates protein structure and function. It is targeted to proteins by lysine acetyltransferases (KATs) or removed by lysine deacetylases. This work identifies a role for the KAT enzyme general control of amino acid synthesis protein 5 (GCN5; KAT2A) in regulating muscle integrity by inhibiting DNA binding of the transcription factor/repressor Yin Yang 1 (YY1). Here we report that a muscle-specific mouse knockout of GCN5 (Gcn5skm-/-) reduces the expression of key structural muscle proteins, including dystrophin, resulting in myopathy. GCN5 was found to acetylate YY1 at two residues (K392 and K393), disrupting the interaction between the YY1 zinc finger region and DNA. These findings were supported by human data, including an observed negative correlation between YY1 gene expression and muscle fiber diameter. Collectively, GCN5 positively regulates muscle integrity through maintenance of structural protein expression via acetylation-dependent inhibition of YY1. This work implicates the role of protein acetylation in the regulation of muscle health and for consideration in the design of novel therapeutic strategies to support healthy muscle during myopathy or aging.

Understanding the Therapeutic Potential of Ascorbic Acid in the Battle to Overcome Cancer.

Cancer, a fatal disease, is also one of the main causes of death worldwide. Despite various developments to prevent and treat cancer, the side effects of anticancer drugs remain a major concern. Ascorbic acid is an essential vitamin required by our bodies for normal physiological function and also has antioxidant and anticancer activity. Although the body cannot synthesize ascorbic acid, it is abundant in nature through foods and other natural sources and also exists as a nutritional food supplement. In anticancer drug development, ascorbic acid has played an important role by inhibiting the development of cancer through various mechanisms, including scavenging reactive oxygen species (ROS), selectively producing ROS and encouraging their cytotoxicity against tumour cells, preventing glucose metabolism, serving as an epigenetic regulator, and regulating the expression of HIF in tumour cells. Several ascorbic acid analogues have been produced to date for their anticancer and antioxidant activity. The current review summarizes the mechanisms behind ascorbic acid's antitumor activity, presents a compilation of its derivatives and their biological activity as anticancer agents, and discusses delivery systems such as liposomes, nanoparticles against cancer, and patents on ascorbic acid as anticancer agents.

Epigallocatechin-3-gallate (EGCG) Alters Histone Acetylation and Methylation and Impacts Chromatin Architecture Profile in Human Endothelial Cells.

Epigallocatechin gallate (EGCG), the main green tea polyphenol, exerts a wide variety of biological actions. Epigenetically, the catechin has been classified as a DNMTs inhibitor, however, its impact on histone modifications and chromatin structure is still poorly understood. The purpose of this study was to find the impact of EGCG on the histone posttranslational modifications machinery and chromatin remodeling in human endothelial cells of both microvascular (HMEC-1) and vein (HUVECs) origin. We analyzed the methylation and acetylation status of histones (Western blotting), as well as assessed the activity (fluorometric assay kit) and gene expression (qPCR) of the enzymes playing a prominent role in shaping the human epigenome. The performed analyses showed that EGCG increases histone acetylation (H3K9/14ac, H3ac), and methylation of both active (H3K4me3) and repressive (H3K9me3) chromatin marks. We also found that the catechin acts as an HDAC inhibitor in cellular and cell-free models. Additionally, we observed that EGCG affects chromatin architecture by reducing the expression of heterochromatin binding proteins: HP1α, HP1γ. Our results indicate that EGCG promotes chromatin relaxation in human endothelial cells and presents a broad epigenetic potential affecting expression and activity of epigenome modulators including HDAC5 and 7, p300, CREBP, LSD1 or KMT2A.

Anacardic acid attenuates pressure-overload cardiac hypertrophy through inhibiting histone acetylases.

Cardiac hypertrophy has become a major cardiovascular problem wordwide and is considered the early stage of heart failure. Treatment and prevention strategies are needed due to the suboptimal efficacy of current treatment methods. Recently, many studies have demonstrated the important role of histone acetylation in myocardium remodelling along with cardiac hypertrophy. A Chinese herbal extract containing anacardic acid (AA) is known to possess strong histone acetylation inhibitory effects. In previous studies, we demonstrated that AA could reverse alcohol-induced cardiac hypertrophy in an animal model at the foetal stage. Here, we investigated whether AA could attenuate cardiac hypertrophy through the modulation of histone acetylation and explored its potential mechanisms in the hearts of transverse aortic constriction (TAC) mice. This study showed that AA attenuated hyperacetylation of acetylated lysine 9 on histone H3 (H3K9ac) by inhibiting the expression of p300 and p300/CBP-associated factor (PCAF) in TAC mice. Moreover, AA normalized the transcriptional activity of the heart nuclear transcription factor MEF2A. The high expression of cardiac hypertrophy-linked genes (ANP, ß-MHC) was reversed through AA treatment in the hearts of TAC mice. Additionally, we found that AA improved cardiac function and survival rate in TAC mice. The current results further highlight the mechanism by which histone acetylation is controlled by AA treatment, which may help prevent and treat hypertrophic cardiomyopathy.

Discovery of 1,8-acridinedione derivatives as novel GCN5 inhibitors via high throughput screening.

The general control nonrepressed protein 5 (GCN5) plays a crucial role in many biological processes. Dysregulation of GCN5 has been closely related to various human diseases, especially cancers. Hence, the exploitation of small molecules targeting GCN5 is essential for drug design and academic research. Based on the amplified luminescent proximity homogeneous assay screen methodology, we performed high throughput screening and discovered a novel GCN5 inhibitor DC_G16 with 1,8-acridinedione scaffold. Structure optimization led to the identification of a highly potent inhibitor, namely DC_G16-11 with the half-maximal inhibitory concentration (IC50) value of 6.8 µM. The binding between DC_G16-11 and GCN5 was demonstrated by NMR and SPR with a KD of 4.2 µM. It could also inhibit proliferation and induce cell cycle arrest and apoptosis in cancer cells while it presented minimal effects on normal cells. Herein, DC_G16-11 could be applied as a validated chemical probe for GCN5-related biological function research and presented great potential for clinical disease treatment.

Therapeutic effect of Cryptotanshinone on experimental rheumatoid arthritis through downregulating p300 mediated-STAT3 acetylation.

BACKGROUND AND PURPOSE: The balance between T helper 17 (Th17) cells and regulatory T (Treg) cells, plays a critical role in rheumatoid arthritis (RA). The differentiation of Th17 cells requires the activation of STAT3, which determines the balance of Th17/Treg. Here, we investigated the therapeutic effect of Cryptotanshinone (CTS) on collagen induced mouse arthritis and explored the underlying mechanisms. EXPERIMENTAL APPROACH: Arthritis was induced in DBA/1 mice with bovine collagen type II and complete Freund's adjuvant. CTS was given at 20mgkg-1d-1 or 60mgkg-1d-1 by gavage for 6weeks. The immuno-inflammation and joint destruction were evaluated and the balance of Th17/Treg was determined. STAT3 acetylation and phosphorylation were detected by western blotting, and the involvement of p300 was investigated by siRNA and plasmid overexpression. KEY RESULTS: CTS at a dose of 60mgkg-1d-1 ameliorated the inflammation and joint destruction in CIA mice. It improved Th17/Treg imbalance, and inhibited both acetylation and phosphorylation of STAT3. CTS reduced p300 expression and its binding to STAT3, but increased phosphorylated AMPK. Knockdown of p300 mimicked the inhibitory effect of CTS on STAT3 acetylation and phosphorylation, which could be partially rescued by overexpression of p300-WT, but not p300-dominant negative (DN) construct. CONCLUSION AND IMPLICATIONS: Our study suggested that the anti-arthritis effects of CTS were attained through suppression of p300-mediated STAT3 acetylation. Our data suggest that CTS might be a potential immune modulator for RA treatment.

Diencephalic Size Is Restricted by a Novel Interplay Between GCN5 Acetyltransferase Activity and Retinoic Acid Signaling.

Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5hat/hat )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5hat/hat ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARß. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5hat/hat ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric disorders. However, the mechanisms that regulate diencephalic morphogenesis and the involvement of RA signaling in this process are poorly understood. Here we demonstrate a novel role of the acetyltransferase GCN5 in a previously undescribed mechanism of RA signaling in the developing forebrain that is required to maintain the appropriate size of the diencephalon. Together, our experiments identify a novel nonhistone substrate of GCN5, highlight an essential role for both GCN5 and RA signaling in early diencephalic development, and elucidate a novel molecular regulatory mechanism for RA signaling that is specific to the developing forebrain.

Ginsenoside Reduces Cognitive Impairment During Chronic Cerebral Hypoperfusion Through Brain-Derived Neurotrophic Factor Regulated by Epigenetic Modulation.

Increased expression of brain-derived neurotrophic factor (BDNF) has been associated with memory-enhancing and neuroprotective properties of some drugs under chronic cerebral hypoperfusion (CCH) condition. Ginsenoside Rd (GSRd), one of the main active ingredients in Panax ginseng, is widely used for brain protection. However, it is poorly understood whether epigenetic mechanisms implied in the BDNF modulation after GSRd treatment for CCH remain elusive. Here, we investigated the neuroprotective effects of GSRd and the involved mechanisms. We demonstrated that GSRd administration ameliorated CCH-induced impairment of learning and memory behaviors, evidenced by decreased escape latency and increased number of crossing the platform in Morris water maze test. This improvement was associated with promoted neuron survival and increased BDNF expression in the hippocampus and prefrontal cortex of CCH mice. GSRd improved neuron survival and decreased neuron apoptosis and the level of caspase-3 under oxygen-glucose deprivation/reoxygenation (OGD/R) by upregulation of BDNF as well as in vitro. The levels of acetylated histone H3 (Ac-H3) and histone deacetylase (histone deacetylase 2 (HDAC2)) were altered under OGD/R in a time-dependent manner, and GSRd reestablished the balance between Ac-H3 and HDAC2 which resulted in upregulation of BDNF and increased neuron survival. MS-275, an inhibitor of class I HDACs, abolished the levels of Ac-H3 at the bdnf promoters and enhanced upregulation of BDNF after GSRd administration, suggesting a synergistic effect between GSRd and MS-275. All the data suggested that GSRd provided neuroprotection by epigenetic modulation which accounted for the regulation of BDNF in CCH mice.

Folate Receptor Alpha Upregulates Oct4, Sox2 and Klf4 and Downregulates miR-138 and miR-let-7 in Cranial Neural Crest Cells.

Prenatal folic acid (FA) supplementation prevents neural tube defects. Folate receptor alpha (FRα) is critical for embryonic development, including neural crest (NC) development. Previously we showed that FRα translocates to the nucleus in response to FA, where it acts as a transcription factor. In this study, we examined if FA through interaction with FRα regulates stem cell characteristics of cranial neural crest cells (CNCCs)-critical for normal development. We hypothesized that FRα upregulates coding genes and simultaneously downregulates non-coding miRNA which targets coding genes in CNCCs. Quantitative RT-PCR and chromatin immunoprecipitation showed that FRα upregulates Oct4, Sox2, and Klf4 by binding to their cis-regulator elements-5' enhancer/promoters defined by H3K27Ac and p300 occupancy. FA via FRα downregulates miRNAs, miR-138 and miR-let-7, which target Oct4 and Trim71 (an Oct4 downstream effector), respectively. Co-immunoprecipitation data suggests that FRα interacts with the Drosha-DGCR8 complex to affect pre-miRNA processing. Transfecting anti-miR-138 or anti-miR-let-7 into non-proliferating neural crest cells (NCCs) derived from Splotch (Sp-/- ), restored their proliferation potential. In summary, these results suggest a novel pleiotropic role of FRα: (a) direct activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules. Stem Cells 2016;34:2721-2732.

Developmental exposure to 50 parts-per-billion arsenic influences histone modifications and associated epigenetic machinery in a region- and sex-specific manner in the adult mouse brain.

Epidemiological studies report that arsenic exposure via drinking water adversely impacts cognitive development in children and, in adults, can lead to greater psychiatric disease susceptibility, among other conditions. While it is known that arsenic toxicity has a profound effect on the epigenetic landscape, very few studies have investigated its effects on chromatin architecture in the brain. We have previously demonstrated that exposure to a low level of arsenic (50ppb) during all three trimesters of fetal/neonatal development induces deficits in adult hippocampal neurogenesis in the dentate gyrus (DG), depressive-like symptoms, and alterations in gene expression in the adult mouse brain. As epigenetic processes control these outcomes, here we assess the impact of our developmental arsenic exposure (DAE) paradigm on global histone posttranslational modifications and associated chromatin-modifying proteins in the dentate gyrus and frontal cortex (FC) of adult male and female mice. DAE influenced histone 3K4 trimethylation with increased levels in the male DG and FC and decreased levels in the female DG (no change in female FC). The histone methyltransferase MLL exhibited a similar sex- and region-specific expression profile as H3K4me3 levels, while histone demethylase KDM5B expression trended in the opposite direction. DAE increased histone 3K9 acetylation levels in the male DG along with histone acetyltransferase (HAT) expression of GCN5 and decreased H3K9ac levels in the male FC along with decreased HAT expression of GCN5 and PCAF. DAE decreased expression of histone deacetylase enzymes HDAC1 and HDAC2, which were concurrent with increased H3K9ac levels but only in the female DG. Levels of H3 and H3K9me3 were not influenced by DAE in either brain region of either sex. These findings suggest that exposure to a low, environmentally relevant level of arsenic during development leads to long-lasting changes in histone methylation and acetylation in the adult brain due to aberrant expression of epigenetic machinery based on region and sex.

[Effects of moxibustion with seed-sized moxa cone on apoptosis of myocardial cells after sport fatigue in mice].

OBJECTIVE: To observe the effects of moxibustion on factors related with apoptosis of myocardial cells after sports fatigue in mice as well as the relationship among histone acetyltransferases p300 (p300), CREB binding protein (CBP) and cell apoptosis to discuss the role of p300 and CBP in moxibustion against apoptosis of myocardial cells. METHODS: Sixty clean-grade male Kunming mice were randomly divided into a control group, a sport group and a moxibustion group, 20 cases in each one. Mice in all group received identical feeding environment. Mice in the control group did not received sport nor moxibustion; mice in the sport group and moxibustion group received non-weight swimming training which lasted from 30 min per day to 90 min per day gradually for 21 days; 1 h after swimming training, mice in the moxibustion group received moxibustion with seed-sized moxa cone at "Zusanli" (ST 36) and "Guanyuan" (CV 4), 5 cones at each acupoint, once a day for 21 days. 24 h after the final swimming training, cardiac muscle tissue was collected to test factor associated suicide (Fas), B cell lymphoma/lewkmia-2 (Bcl-2) by immunohistochemical method and expression of p300 and CBP. RESULTS: Compared with the control group, the apoptosis rate of myocardial cells in the sport group was significantly increased (P<0.01), and apoptosis body with dense distribution and deep coloring can be seen in the field of microscope; the expression of Fas protein was significantly increased (P<0.01), and expression of Bcl-2, p300 and CBP was reduced (all P<0.01). The equally distributed apoptosis body with slight coloring was seen in the moxibustion group. Compared with the sport group, the apoptosis rate of myocardial cells in the moxibustion group was significantly reduced (P<0.05); the expression of Fas protein was significantly reduced (P<0.05), and expression of Bcl-2, p300 and CBP was increased (all P<0.05). CONCLUSION: Moxibustion could promote the expression of p300 and CBP in myocardial cells after sports fatigue in mice to inhibit the starting of apoptotic process, therefore reducing the apoptosis of myocardial cells after heavy exercise and protecting heart function.

Gallic Acid Decreases Inflammatory Cytokine Secretion Through Histone Acetyltransferase/Histone Deacetylase Regulation in High Glucose-Induced Human Monocytes.

Hyperglycemia contributes to diabetes and several diabetes-related complications. Gallic acid is a polyhydroxy phenolic compound found in various natural products. In this study, we investigated the effects and mechanism of gallic acid on proinflammatory cytokine secretion in high glucose-induced human monocytes (THP-1 cells). THP-1 cells were cultured under normoglycemic or hyperglycemic conditions, in the absence or presence of gallic acid. Hyperglycemic conditions significantly induced histone acetylation, nuclear factor-κB (NF-κB) activation, and proinflammatory cytokine release from THP-1 cells, whereas gallic acid suppressed NF-κB activity and cytokine release. It also significantly reduced CREB-binding protein/p300 (CBP/p300, a NF-κB coactivator) gene expression, acetylation levels, and CBP/p300 histone acetyltransferase (HAT) activity. In addition, histone deacetylase 2 (HDAC2) expression was significantly induced. These results suggest that gallic acid inhibits hyperglycemic-induced cytokine production in monocytes through epigenetic changes involving NF-κB. Therefore, gallic acid may have potential for the treatment and prevention of diabetes and its complications.

Yin-Yang strands of PCAF/Hedgehog axis in cancer control.

PCAF (p300/CBP associated factor) harbors acetyltransferase and a recently identified ubiquitylation activity that regulates gene expression in response to genotoxic stress or mitogenic signals. We highlight the dual role of PCAF in the control of Hedgehog signaling, a master regulator of tissue development, stemness, and tumorigenesis. By promoting histone acetylation at Hedgehog/GLI1 target gene promoters or direct ubiquitylation and proteolysis of GLI1, the PCAF/GLI1 axis stands as a promising therapeutic target for Hedgehog-dependent tumors.

Zyflamend, a polyherbal mixture, down regulates class I and class II histone deacetylases and increases p21 levels in castrate-resistant prostate cancer cells.

BACKGROUND: Zyflamend, a mixture containing extracts of ten herbs, has shown promise in a variety of preclinical cancer models, including prostate cancer. The current experiments were designed to investigate the effects of Zyflamend on the expression of class I and II histone deacetylases, a family of enzymes known to be over expressed in a variety of cancers. METHODS: CWR22Rv1 cells, a castrate-resistant prostate cancer cell line, were treated with Zyflamend and the expression of class I and II histone deacetylases, along with their downstream target the tumor suppressor gene p21, was investigated. Involvement of p21 was confirmed with siRNA knockdown and over expression experiments. RESULTS: Zyflamend down-regulated the expression of all class I and II histone deacetylases where Chinese goldthread and baikal skullcap (two of its components) appear to be primarily responsible for these results. In addition, Zyflamend up regulated the histone acetyl transferase complex CBP/p300, potentially contributing to the increase in histone 3 acetylation. Expression of the tumor suppressor gene p21, a known downstream target of histone deacetylases and CBP/p300, was increased by Zyflamend treatment and the effect on p21 was, in part, mediated through Erk1/2. Knockdown of p21 with siRNA technology attenuated Zyflamend-induced growth inhibition. Over expression of p21 inhibited cell growth and concomitant treatment with Zyflamend enhanced this effect. CONCLUSIONS: Our results suggest that the extracts of this polyherbal combination increase histone 3 acetylation, inhibit the expression of class I and class II histone deacetylases, increase the activation of CBP/p300 and inhibit cell proliferation, in part, by up regulating p21 expression.

Measuring CREB activation using bioluminescent probes that detect KID-KIX interaction in living cells.

The cyclic adenosine monophosphate response element-binding protein (CREB) is a transcription factor that contributes to memory formation. The transcriptional activity of CREB is induced by its phosphorylation at Ser-133 and subsequent interaction with the CREB-binding protein (CBP)/p300. We designed and optimized firefly split luciferase probe proteins that detect the interaction of the kinase-inducible domain (KID) of CREB and the KIX domain of CBP/p300. The increase in the light intensity of the probe proteins results from the phosphorylation of the responsible serine corresponding to Ser-133 of CREB. Because these proteins have a high signal-to-noise ratio and are nontoxic, it has become possible for the first time to carry out long-term measurement of KID-KIX interaction in living cells. Furthermore, we examined the usefulness of the probe proteins for future high-throughput cell-based drug screening and found several herbal extracts that activated CREB.

A conserved cysteine cluster, essential for transcriptional activity, mediates homodimerization of human metal-responsive transcription factor-1 (MTF-1).

Metal-responsive transcription factor-1 (MTF-1) is a zinc finger protein that activates transcription in response to heavy metals such as Zn(II), Cd(II) and Cu(I) and is also involved in the response to hypoxia and oxidative stress. MTF-1 recognizes a specific DNA sequence motif termed the metal response element (MRE), located in the promoter/enhancer region of its target genes. The functional domains of MTF-1 include, besides the DNA-binding and activation domains and signals for subcellular localization (NLS and NES), a cysteine cluster 632CQCQCAC638 located near the C-terminus. Here we show that this cysteine cluster mediates homodimerization of human MTF-1, and that dimer formation in vivo is important for basal and especially metal-induced transcriptional activity. Neither nuclear translocation nor DNA binding is impaired in a mutant protein in which these cysteines are replaced by alanines. Although zinc supplementation induces MTF-1 dependent transcription it does not per se enhance dimerization, implying that actual zinc sensing is mediated by another domain. By contrast copper, which on its own activates MTF-1 only weakly in the cell lines tested, stabilizes the dimer by inducing intermolecular disulfide bond formation and synergizes with zinc to boost MTF-1 dependent transcription.

Low-molecular-weight compounds having neurotrophic activity in cultured PC12 cells and neurons.

Recent reports have indicated that some low-molecular-weight compounds mimic neurotrophic factors inducing neurite outgrowth and neuroprotection. Carnosic acid (CA) promotes neurite outgrowth through the activation of Nrf2 in PC12 cells. CA also protects neurons via the keap/Nrf2 transcriptional pathway from oxidative stress. Forskolin-induced neurite outgrowth is mediated by activation of the PKA signalling pathway and this PKA-mediated neurite outgrowth is achieved by the expression of nur77 in PC12 cells. In addition, forskolin at its low concentration is closely related to the cAMP-induced protective function against L-DOPA-induced cytotoxicity in PC12 cells. A HDAC inhibitor trichostatin A (TSA) increases neurite length via p53 acetylation in rat cultured cerebellar granule neurons and in cerebral cortical neurons, and also protects neurons against glutathione depletion-induced oxidative stress. Recently, it was revealed that Nrf2 and p53 bind to CBP/p300 directly, and Nur77 is acetylated in vivo and in vitro by CBP/p300. Acetylation of Nrf2, p53 and Nur77 by CBP/p300 may constitute a novel similar regulatory mechanism for low-molecular-weight compounds with neurotrophic activities.

Effect of artemisinin combined with glucocorticoid on the expressions of glucocorticoid receptor α mRNA, glucocorticoid receptor ß mRNA and P300/CBP protein in lupus nephritis mice.

OBJECTIVE: To investigate the therapeutic effects and mechanisms of using artemisinin (Art) combined with glucocorticoid (GC) to treat lupus nephritis (LN) mice. METHODS: Forty hybrid female mice were randomly and equally divided into four groups with the method of random number table: control group, model group, prednisone group administrated with 6.45 mg/(kg·d) prednisone suspension, and Art+prednisone group administrated with 150 mg/(kg·d) Art suspension and 3.225 mg/(kg·d) prednisone suspension. A mice model of LN was established by injection with living lymph cell suspension. The changes of urine protein/24h, the expressions of GC receptor α (GRα) mRNA, GC receptor ß (GRß) mRNA in peripheral blood mononuclear cells (PBMCs), and transcriptional coactivator P300/CBP protein in renal tissue were measured. RESULTS: Compared with the model group, the treatment groups had significant decrease in urine protein/24 h, and renal pathological lesion (P<0.01). In the same groups, the expression of transcriptional coactivator P300/CBP protein in renal tissue and GRα mRNA were significantly increased, and GRß mRNA expression was significantly decreased (P<0.01). And the Art+prednisone group has a better therapeutic effect than the prednisone group (P<0.01). CONCLUSIONS: Art has therapeutic sensitization effects on GC in the LN mice. The underlying mechanism could be correlated with the effect of Art on the increase of the expressions of GRα mRNA and transcriptional coactivator P300 300/CBP protein in renal tissue and on the decrease of the expression of GRß mRNA in PBMC.

Mutant hypoxia inducible factor-1α improves angiogenesis and tissue perfusion in ischemic rabbit skeletal muscle.

Hypoxia-inducible factor-1α (HIF-1α) is one of the most potent angiogenic growth factors. It regulates genes involved in angiogenesis, but is inactivated rapidly by normoxia. Ad-HIF-1α-Trip was constructed by transforming Pro402, Pro564, and Asn803 in HIF-1α to alanine in order to delay degradation and create a constitutive transcriptional activator. In this study, we investigated whether Ad-HIF-1α-Trip could induce functional mature angiogenesis and the possible mechanisms involved. We found that Ad-HIF-1α-Trip increased the expression of multiple angiogenic genes in cultured HMVEC-Ls, including VEGF, PLGF, PAI-1, and PDGF. In a rabbit model of acute hind limb ischemia, Ad-HIF-1α-Trip improved tissue perfusion and collateral vessels, as measured by contrast-enhanced ultrasound (CEU), CT angiography, and vascular casting. Ad-HIF-1α-Trip also produced more histologically identifiable capillaries, which were verified by immunostaining, compared with controls. Interestingly, inhibition of CBP/p300 by curcumin prevented HIF-1α from inducing the expression of several angiogenic genes. The present study suggests that Ad-HIF-1α-Trip can induce mature angiogenesis and improve tissue perfusion in ischemic rabbit skeletal muscle. CBP/p300, which interacts with the transactivation domains of HIF-1α, is important for HIF-1α-induced transcription of angiogenic genes.

Histone modifiers, YY1 and p300, regulate the expression of cartilage-specific gene, chondromodulin-I, in mesenchymal stem cells.

Elucidating the regulatory mechanism for tissue-specific gene expression is key to understanding the differentiation process. The chondromodulin-I gene (ChM-I) is a cartilage-specific gene, the expression of which is regulated by the transcription factor, Sp3. The binding of Sp3 to the core-promoter region is regulated by the methylation status of the Sp3-binding motif as we reported previously. In this study, we have investigated the molecular mechanisms of the down-regulation of ChM-I expression in mesenchymal stem cells (MSCs) and normal mesenchymal tissues other than cartilage. The core-promoter region of cells in bone and peripheral nerve tissues was hypermethylated, whereas the methylation status in cells of other tissues including MSCs did not differ from that in cells of cartilage, suggesting the presence of inhibitory mechanisms other than DNA methylation. We found that a transcriptional repressor, YY1, negatively regulated the expression of ChM-I by recruiting histone deacetylase and thus inducing the deacetylation of associated histones. As for a positive regulator, we found that a transcriptional co-activator, p300, bound to the core-promoter region with Sp3, inducing the acetylation of histone. Inhibition of YY1 in combination with forced expression of p300 and Sp3 restored the expression of ChM-I in cells with a hypomethylated promoter region, but not in cells with hypermethylation. These results suggested that the expression of tissue-specific genes is regulated in two steps; reversible down-regulation by transcriptional repressor complex and tight down-regulation via DNA methylation.