Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix

Apoptosis of VSMCs (vascular smooth-muscle cells) leads to features of atherosclerotic plaque instability. We have demonstrated previously that plaque-derived VSMCs have reduced IGF1 (insulin-like growth factor 1) signalling, resulting from a decrease in the expression of IGF1R (IGF1 receptor) compared with normal aortic VSMCs [Patel, Zhang, Siddle, Soos, Goddard, Weissberg and Bennett (2001) Circ. Res. 88, 895-902]. In the present study, we show that apoptosis induced by oxidative stress is inhibited by ectopic expression of IGF1R. Oxidative stress repressed IGF1R expression at multiple levels, and this was also blocked by mutant p53. Oxidative stress also induced p53 phosphorylation and apoptosis in VSMCs. p53 negatively regulated IGF1R promoter activity and expression and, consistent with this, p53-/- VSMCs demonstrated increased IGF1R expression, both in vitro and in advanced atherosclerotic plaques in vivo. Oxidative-stress-induced interaction of endogenous p53 with TBP (TATA-box-binding protein) was dependent on p53 phosphorylation. Oxidative stress also increased the association of p53 with HDAC1 (histone deacetylase 1). Trichostatin A, a specific HDAC inhibitor, or p300 overexpression relieved the repression of IGF1R following oxidative stress. Furthermore, acetylated histone-4 association with the IGF1R promoter was reduced in cells subjected to oxidative stress. These results suggest that oxidative-stress-induced repression of IGF1R is mediated by the association of phosphorylated p53 with the IGF1R promoter via TBP, and by the subsequent recruitment of chromatin-modifying proteins, such as HDAC1, to the IGF1R promoter-TBP-p53 complex.     

High levels of glucose induce “metabolic memory” in cardiomyocyte via epigenetic histone H3 lysine 9 methylation

Diabetic patients continue to develop inflammation and cardiovascular complication even after achieving glycemic control, suggesting a "metabolic memory". Metabolic memory is a major challenge in the treatment of diabetic complication, and the mechanisms underlying metabolic memory are not clear. Recent studies suggest a link between chromatin histone methylation and metabolic memory. In this study, we tested whether histone 3 lysine-9 tri-methylation (H3K9me3), a key epigenetic chromatin marker, was involved in high glucose (HG)-induced inflammation and metabolic memory. Incubating cardiomyocyte cells in HG resulted in increased levels of inflammatory cytokine IL-6 mRNA when compared with myocytes incubated in normal culture media, whereas mannitol (osmotic control) has no effect. Chromatin immunoprecipitation (ChIP) assays showed that H3K9me3 levels were significantly decreased at the promoters of IL-6. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase, Suv39h1, were also reduced after HG treatment. HG-induced apoptosis, mitochondrial dysfunction and cytochrome-c release were reversible. However, the effects of HG on the expression of IL-6 and the levels of H3K9me3 were irreversible after the removal of HG from the culture. These results suggest that HG-induced sustained inflammatory phenotype and epigenetic histone modification, rather than HG-induced mitochondrial dysfunction and apoptosis, are main mechanisms responsible for metabolic memory. In conclusion, our data demonstrate that HG increases expression of inflammatory cytokine and decreases the levels of histone-3 methylation at the cytokine promoter, and suggest that modulating histone 3 methylation and inflammatory cytokine expression may be a useful strategy to prevent metabolic memory and cardiomyopathy in diabetic patients.     

Retinoic acid receptor-mediated signaling protects cardiomyocytes from hyperglycemia induced apoptosis: role of the renin-angiotensin system

Diabetes mellitus (DM) is a primary risk factor for cardiovascular diseases and heart failure. Activation of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) has an anti-diabetic effect; but, a role in diabetic cardiomyopathy remains unclear. Using neonatal and adult cardiomyocytes, we determined the role of RAR and RXR in hyperglycemia-induced apoptosis and expression of renin-angiotensin system (RAS) components. Decreased nuclear expression of RARα and RXRα, activation of apoptotic signaling and cell apoptosis was observed in high glucose (HG) treated neonatal and adult cardiomyocytes and diabetic hearts in Zucker diabetic fatty (ZDF) rats. HG-induced apoptosis and reactive oxygen species (ROS) generation was prevented by both RAR and RXR agonists. Silencing expression of RARα and RXRα, by small interference RNA, promoted apoptosis under normal conditions and significantly enhanced HG-induced apoptosis, indicating that RARα and RXRα are required in regulating cell apoptotic signaling. Blocking angiotensin type 1 receptor (AT(1) R); but, not AT(2) R, attenuated HG-induced apoptosis and ROS generation. Moreover, HG induced gene expression of angiotensinogen, renin, AT(1) R, and angiotensin II (Ang II) synthesis were inhibited by RARα agonists and promoted by silencing RARα. Activation of RXRα, downregulated the expression of AT(1) R; and RXRα silencing accelerated HG induced expression of angiotensinogen and Ang II synthesis, whereas there was no significant effect on renin gene expression. These results indicate that reduction in the expression of RARα and RXRα has an important role in hyperglycemia mediated apoptosis and expression of RAS components. Activation of RAR/RXR signaling protects cardiomyocytes from hyperglycemia, by reducing oxidative stress and inhibition of the RAS.     

Low expression of miR-186-5p regulates cell apoptosis by targeting toll-like receptor 3 in high glucose–induced cardiomyocytes

To investigate the effect and mechanism of microRNA-186-5p (miR-186-5p) on the apoptosis in high glucose (HG)–treated cardiomyocytes. Diabetic cardiomyopathy model was established in cardiomyocytes by stimulating with HG. The expressions of miR-186-5p and toll-like receptor 3 (TLR3) were detected by quantitative polymerase chain reaction or Western blot analysis, respectively. Apoptosis was detected in HG-treated cardiomyocytes by flow cytometry and Western blot analysis. The interaction between miR-186-5p and TLR3 was explored by bioinformatics analysis and luciferase activity assay. Results showed that miR-186-5p expression was downregulated in HG-treated cardiomyocytes and its overexpression reversed HG-induced apoptosis and cleaved caspase-3 protein expression. Moreover, TLR3 was indicated as a target of miR-186-5p and regulated by miR-186-5p. Knockdown of TLR3 suppressed HG-induced apoptosis and cleaved caspase-3 protein expression. Besides, restoration of TLR3 ablated the effect of miR-186-5p on cell apoptosis. Collectively, miR-186-5p attenuated HG-induced apoptosis by regulating TLR3 in cardiomyocytes, providing novel biomarker for treatment of diabetic cardiomyopathy.     

Hyperglycemia induces apoptosis via CB1 activation through the decrease of FAAH 1 in retinal pigment epithelial cells

Fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the main endocannabinoid, anandamide, and related fatty acid amides, has emerged as a regulator of endocannabinoid signaling. Retinal pigment epithelial (RPE) cells are believed to be important cells in the pathogenesis of diabetic retinopathy. However, the pathophysiology of FAAH in diabetic retinopathy has not been determined. Thus, we examined the effect of high glucose (HG) on the expression of FAAH and CB(1)R in the ARPE-19 human RPE cells. We found that HG downregulated the expression of FAAH 1 mRNA and protein in ARPE-19 cells. In contrast, it upregulated the expression of CB(1)R mRNA and protein. HG-induced internalization of CB(1)R in HEK 293 cells and ARPE-19 cells was blocked by overexpression of FAAH 1 and treatment with the CB(1)R blocker, AM 251. HG-induced generation of reactive oxygen species and lipid peroxide formation were blocked by the overexpression of FAAH 1. FAAH 1 overexpression also blocked HG-induced expression of CB(1)R in the cytosolic fraction. We also investigated whether the overexpression of FAAH 1 protected against HG-induced apoptosis. High glucose increased the Bax/Bcl-2 ratio and levels of cleaved PARP, cleaved caspase-9 and caspase-3, and reduced cell viability. HG-induced apoptotic effects were reduced by the overexpression of FAAH 1, treatment with the CB(1)R-specific antagonist AM 251 and CB(1)R siRNA transfection. In conclusion, HG-induced apoptosis in ARPE-19 cells by inducing CB(1)R expression through the downregulation of FAAH 1 expression. Our results provide evidence that CB(1)R blockade through the recovery of FAAH 1 expression may be a potential anti-diabetic therapy for the treatment of diabetic retinopathy.     

MALAT1/miR-185-5p mediated high glucose-induced oxidative stress,mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway

To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR-185-5p with MALAT1 and RhoA were validated by dual-luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D-glucose (HG) in the presence or absence of MALAT1-shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR-185-5p expression were determined by real-time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1^S616, mitofusin 2 and apoptosis-related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC-1. MALAT1 was significantly up-regulated while miR-185-5p was down-regulated in myocardium of db/db mice and HG-induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG-induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p and mediated HG-induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.     

High glucose-induced repression of RAR/RXR in cardiomyocytes is mediated through oxidative stress/JNK signaling

The biological actions of retinoids are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We have recently reported that decreased expression of RARα and RXRα has an important role in high glucose (HG)-induced cardiomyocyte apoptosis. However, the regulatory mechanisms of HG effects on RARα and RXRα remain unclear. Using neonatal cardiomyocytes, we found that ligand-induced promoter activity of RAR and RXR was significantly suppressed by HG. HG promoted protein destabilization and serine-phosphorylation of RARα and RXRα. Proteasome inhibitor MG132 blocked the inhibitory effect of HG on RARα and RXRα. Inhibition of intracellular reactive oxidative species (ROS) abolished the HG effect. In contrast, H(2)O(2) stimulation suppressed the expression and ligand-induced promoter activity of RARα and RXRα. HG promoted phosphorylation of ERK1/2, JNK and p38 MAP kinases, which was abrogated by an ROS inhibitor. Inhibition of JNK, but not ERK and p38 activity, reversed HG effects on RARα and RXRα. Activation of JNK by over expressing MKK7 and MEKK1, resulted in significant downregulation of RARα and RXRα. Ligand-induced promoter activity of RARα and RXRα was also suppressed by overexpression of MEKK1. HG-induced cardiomyocyte apoptosis was potentiated by activation of JNK, and prevented by all-trans retinoic acid and inhibition of JNK. Silencing the expression of RARα and RXRα activated the JNK pathway. In conclusion, HG-induced oxidative stress and activation of the JNK pathway negatively regulated expression/activation of RAR and RXR. The impaired RAR/RXR signaling and oxidative stress/JNK pathway forms a vicious circle, which significantly contributes to hyperglycemia induced cardiomyocyte apoptosis.     

Mutant p53 binds to estrogen receptor negative promoter via DNMT1 and HDAC1 in MDA-MB-468 breast cancer cells

DNA methylation and histone deacetylation are two epigenetic mechanisms involved in the lack of estrogen receptor (ER) expression. Our previous studies demonstrated that mutant p53 along with repression complex proteins including DNMT1, HDAC1 and MeCP2 is associated with ER-negative promoter in MDA-MB-468 cells. To elucidate the molecular mechanism of estrogen receptor 1 (ESR1) gene silencing in these cells, we down-regulated DNMT1 and HDAC1 expression using siRNAs and studied the ability of DNMT1, HDAC1, MeCP2 and p53 in binding to ESR1 promoter CpG island. Our results showed that DNMT1 or HDAC1 down-regulation disassembled the repression complex proteins and mutant p53 from ER-negative promoter. The partial demethylation of ESR1 promoter and ER re-expression in down-regulated cells supports these findings. In vivo binding studies demonstrated that mutation of p53 protein in this cell line did not affect its binding capacity to DNMT1, HDAC1 and MeCP2 proteins. Our observations suggest that not only histone deacetylase activity of HDAC1 contributes to inactivation of methylated ESR1 gene but also HDAC1 presence on ESR1 promoter is important for assembly of DNMT1 in repression complex. In addition, our data revealed that mutant p53 protein binds to the promoter of ESR1 through direct interaction with HDAC1 and indirect interaction with DNMT1, MeCP2 proteins in the ER-negative MDA-MB-468 cells.     

Rosiglitazone attenuates NF-κB-mediated Nox4 upregulation in hyperglycemia-activated endothelial cells

Vascular complications, a major cause of morbidity and mortality in diabetic patients, are related to hyperglycemia-induced oxidative stress. Previously, we reported that rosiglitazone (RSG) attenuated vascular expression and activity of NADPH oxidases in diabetic mice. The mechanisms underlying these effects remain to be elucidated. We hypothesized that RSG acts directly on endothelial cells to modulate vascular responses in diabetes. To test this hypothesis, human aortic endothelial cells (HAECs) were exposed to normal glucose (NG; 5.6 mmol/l) or high glucose (HG; 30 mmol/l) concentrations. Select HAEC monolayers were treated with RSG, caffeic acid phenethyl ester (CAPE), diphenyleneiodonium (DPI), small interfering (si)RNA (to NF-κB/p65 or Nox4), or Tempol. HG increased the expression and activity of the NADPH oxidase catalytic subunit Nox4 but not Nox1 or Nox2. RSG attenuated HG-induced NF-κB/p65 phosphorylation, nuclear translocation, and binding to the Nox4 promoter. Inhibiting NF-κB with CAPE or siNF-κB/p65 also reduced HG-induced Nox4 expression and activity. HG-induced H(2)O(2) production was attenuated by siRNA-mediated knockdown of Nox4, and HG-induced HAEC monocyte adhesion was attenuated by treatment with RSG, DPI, CAPE, or Tempol. These results indicate that HG exposure stimulates HAEC NF-κB activation, Nox4 expression, and H(2)O(2) production and that RSG attenuates HG-induced oxidative stress and subsequent monocyte-endothelial interactions by attenuating NF-κB/p65 activation and Nox4 expression. This study provides novel insights into mechanisms by which the thiazolidinedione peroxisome proliferator-activated receptor-γ ligand RSG favorably modulates endothelial responses in the diabetic vasculature.     

High level glucose increases mutagenesis in human lymphoblastoid cells

Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. We studied whether high level of glucose (HG) treatment that mimic the hyperglycemic condition in diabetes mellitus is mutagenic. Mutagenesis studies were carried out at both hypoxanthine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Role of p53 in HG-induced mutagenesis was also investigated by using human lymphoblastoid cell lines derived from same donor but differs in p53 statuses; TK6 has wild-type p53, NH32 has null p53, and WTK1 has mutant p53 (ile237). In addition, we studied the influence of antioxidant treatment on HG-induced mutagenesis. Mutation fractions at both loci increased significantly in all three lines at 21 and 28 days after HG treatments. At tk locus, the increase of a class of mutants with normal growth rate is mainly responsible for the overall increased mutant fraction. Compared to TK6 cells, both NH32 and WTK1 cells showed an early onset of mutagenesis. Treatment of cells with antioxidant N-acetyl-L-cysteine partially reduced HG induced mutagenesis. This study is the first to indicate that HG is able to induce gene mutation which may be one of the important mechanisms of diabetes-associated carcinogenesis.     

A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

Insulin-like growth factor 1 receptor (IGF-1R) is important in cancer cell growth and survival and has been implicated in cancer pathophysiology and treatment. Here we report a novel function for IGF-1R in p53-dependent apoptotic response. We show that inhibition or loss of IGF-1R activity reduces translational synthesis of p53 and Mdm2 protein. Notably, IGF-1R inhibition increases p53 protein stability by reducing p53 ubiquitination and maintains p53 at low levels by decreasing p53 synthesis, thus rendering p53 insensitive to stabilization after DNA damage. The accumulation and apoptosis of DNA-damage-induced p53 is therefore reduced in Igf-1r(-/-) mouse embryonic fibroblasts or tumor cells treated with the IGF-1R inhibitor. Furthermore, we find that inhibition of IGF-1R reduces p53 and Mdm2 translation through a gene-specific mechanism mediated by the respective 5' untranslated region of p53 and mdm2 messenger RNA. The eukaryotic translation initiation factor 4F complex is also involved in this translational inhibition. These results demonstrate an unexpected role for translational control by IGF-1R in p53-mediated apoptosis.     

H3 relaxin inhibits the collagen synthesis via ROS‐ and P2X7R‐mediated NLRP3 inflammasome activation in cardiac fibroblasts under high glucose

Excessive production of reactive oxygen species (ROS) and P2X7R activation induced by high glucose increases NLRP3 inflammasome activation, which contributes to the pathogenesis of diabetic cardiomyopathy. Although H3 relaxin has been shown to inhibit cardiac fibrosis induced by isoproterenol, the mechanism has not been well studied. Here, we demonstrated that high glucose (HG) induced the collagen synthesis by activation of the NLRP3 inflammasome, leading to caspase‐1 activation, interleukin‐1β (IL‐1β) and IL‐18 secretion in neonatal rat cardiac fibroblasts. Moreover, we used a high‐glucose model with neonatal rat cardiac fibroblasts and showed that the activation of ROS and P2X7R was augmented and that ROS‐ and P2X7R‐mediated NLRP3 inflammasome activation was critical for the collagen synthesis. Inhibition of ROS and P2X7R decreased NLRP3 inflammasome‐mediated collagen synthesis, similar to the effects of H3 relaxin. Furthermore, H3 relaxin reduced the collagen synthesis via ROS‐ and P2X7R‐mediated NLRP3 inflammasome activation in response to HG. These results provide a mechanism by which H3 relaxin alleviates NLRP3 inflammasome‐mediated collagen synthesis through the inhibition of ROS and P2X7R under HG conditions and suggest that H3 relaxin represents a potential drug for alleviating cardiac fibrosis in diabetic cardiomyopathy.     

Knockdown of FOXO6 inhibits high glucose–induced oxidative stress and apoptosis in retinal pigment epithelial cells

Oxidative stress and apoptosis in retinal pigment epithelium cells are involved in the pathogenesis of diabetic retinopathy (DR). Forkhead box class O 6 (FOXO6) is a member of the FOXO family that can regulate diabetes-induced oxidative stress. However, the role of FOXO6 in DR has not been clarified. The aim of the present study was to investigate the effects of FOXO6 on high glucose (HG)-induced oxidative stress and apoptosis in ARPE-19 cells. The results showed that FOXO6 was overexpressed in clinical vitreous samples from DR patients and in HG-induced ARPE-19 cells. Knockdown of FOXO6 by small interfeing RNA targeting FOXO6 (si-FOXO6) mitigated the HG-induced the production of reactive oxygen species and malondialdehyde, as well as the inhibition of superoxide dismutase activity. Knockdown of FOXO6 reduced the rate of cell apoptosis in HG-induced ARPE-19 cells. The increase in bax expression and decrease in bcl-2 expression caused by HG stimulation were reversed by si-FOXO6 transfection. Furthermore, knockdown of FOXO6 enhanced the activation of Akt/Nrf2 pathway in HG-stimulated ARPE-19 cells. Taken together, suppression of FOXO6 protects ARPE-19 cells from HG-induced oxidative stress and apoptosis, which is in part mediated by the activation of Akt/Nrf2 pathway.     

AMP-activated Protein Kinase (AMPK) Negatively Regulates Nox4-dependent Activation of p53 and Epithelial Cell Apoptosis in Diabetes

Diabetes and high glucose (HG) increase the generation of NADPH oxidase-derived reactive oxygen species and induce apoptosis of glomerular epithelial cells (podocytes). Loss of podocytes contributes to albuminuria, a major risk factor for progression of kidney disease. Here, we show that HG inactivates AMP-activated protein kinase (AMPK), up-regulates Nox4, enhances NADPH oxidase activity, and induces podocyte apoptosis. Activation of AMPK blocked HG-induced expression of Nox4, NADPH oxidase activity, and apoptosis. We also identified the tumor suppressor protein p53 as a mediator of podocyte apoptosis in cells exposed to HG. Inactivation of AMPK by HG up-regulated the expression and phosphorylation of p53, and p53 acted downstream of Nox4. To investigate the mechanism of podocyte apoptosis in vivo, we used OVE26 mice, a model of type 1 diabetes. Glomeruli isolated from these mice showed decreased phosphorylation of AMPK and enhanced expression of Nox4 and p53. Pharmacologic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-riboside in OVE26 mice attenuated Nox4 and p53 expression. Administration of 5-aminoimidazole-4-carboxamide-1-riboside also prevented renal hypertrophy, glomerular basement thickening, foot process effacement, and podocyte loss, resulting in marked reduction in albuminuria. Our results uncover a novel function of AMPK that integrates metabolic input to Nox4 and provide new insight for activation of p53 to induce podocyte apoptosis. The data indicate the potential therapeutic utility of AMPK activators to block Nox4 and reactive oxygen species generation and to reduce urinary albumin excretion in type 1 diabetes.