O-GlcNAcylation-induced GSK-3ß activation deteriorates pressure overload-induced heart failure via lack of compensatory cardiac hypertrophy in mice.

O-GlcNAc transferase (OGT) modulates many functions of proteins via O-GlcNAcylation that adds O-linked ß-N-acetylglucosamine (O-GlcNAc) to the serine/threonine residues of proteins. However, the role of O-GlcNAcylation in cardiac remodeling and function is not fully understood. To examine the effect of O-GlcNAcylation on pressure overload-induced cardiac hypertrophy and subsequent heart failure, transverse aortic constriction (TAC) surgery was performed in wild type (WT) and Ogt transgenic (Ogt-Tg) mice. Four weeks after TAC (TAC4W), the heart function of Ogt-Tg mice was significantly lower than that of WT mice (reduced fractional shortening and increased ANP levels). The myocardium of left ventricle (LV) in Ogt-Tg mice became much thinner than that in WT mice. Moreover, compared to the heart tissues of WT mice, O-GlcNAcylation of GSK-3ß at Ser9 was increased and phosphorylation of GSK-3ß at Ser9 was reduced in the heart tissues of Ogt-Tg mice, resulting in its activation and subsequent inactivation of nuclear factor of activated T cell (NFAT) activity. Finally, the thinned LV wall and reduced cardiac function induced by TAC4W in Ogt-Tg mice was reversed by the treatment of a GSK-3ß inhibitor, TDZD-8. These results imply that augmented O-GlcNAcylation exacerbates pressure overload-induced heart failure due to a lack of compensatory cardiac hypertrophy via O-GlcNAcylation of GSK-3ß, which deprives the phosphorylation site of GSK-3ß to constantly inactivate NFAT activity to prevent cardiac hypertrophy. Our findings may provide a new therapeutic strategy for cardiac hypertrophy and subsequent heart failure.

Fluctuation in O-GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser621 phosphorylation.

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621 Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621 These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.

Phospholamban Is Downregulated by pVHL-Mediated Degradation through Oxidative Stress in Failing Heart.

The E3 ubiquitin ligase, von Hippel-Lindau (VHL), regulates protein expression by polyubiquitination. Although the protein VHL (pVHL) was reported to be involved in the heart function, the underlying mechanism is unclear. Here, we show that pVHL was upregulated in hearts from two types of genetically dilated cardiomyopathy (DCM) mice models. In comparison with the wild-type mouse, both DCM mice models showed a significant reduction in the expression of phospholamban (PLN), a potent inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase, and enhanced interaction between pVHL and PLN. To clarify whether pVHL is involved in PLN degradation in failing hearts, we used carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial membrane potential (MMP)-lowering reagent, to mimic the heart failure condition in PLN-expressing HEK293 cells and found that CCCP treatment resulted in PLN degradation and increased interaction between PLN and pVHL. However, these effects were reversed with the addition of N-acetyl-l-cysteine. Furthermore, the co-transfection of VHL and PLN in HEK293 cells decreased PLN expression under oxidative stress, whereas knockdown of VHL increased PLN expression both under normal and oxidative stress conditions. Together, we propose that oxidative stress upregulates pVHL expression to induce PLN degradation in failing hearts.

Autophagy Deficiency Diminishes Indomethacin-Induced Intestinal Epithelial Cell Damage through Activation of the ERK/Nrf2/HO-1 Pathway.

Nonsteroidal anti-inflammatory drugs (NSAIDs) can cause epithelial cell damage in the stomach, intestine, and colon. NSAIDs are reported to induce autophagy and apoptosis in intestinal epithelial cells; however, their role in cell damage is poorly understood. To examine the role of autophagy in cell damage, we used autophagy-related gene Atg5-conditional knockout mice, in which the Atg5 gene is only knocked out in intestinal epithelial cells. In an indomethacin (IM)-induced gastrointestinal ulcer mouse model, intestinal epithelium damage was reduced in Atg5-conditional knockout mice compared with wild-type mice. IM-induced damage in IEC6 rat intestinal epithelial cells was reduced when Atg5 was silenced (IEC6shAtg5 cells). Western blot analyses indicated that IM-induced apoptosis decreased, and the potent, oxidative stress-related extracellular signal-regulated kinase (ERK)/nuclear factor-erythroid2-like2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway was upregulated in IEC6shAtg5 cells. An experiment using a reactive oxygen species (ROS)-sensitive fluorescent dye in IEC6shAtg5 cells revealed that the amount of ROS at the baseline and the rate of increase after IM treatment were lower than in intact IEC6 cells. The mitochondrial membrane potential at the baseline and the reduction rate in IM-treated IEC6shAtg5 cells were lower than in intact IEC6 cells, indicating that autophagy deficiency increased ROS production caused by mitochondrial disturbance. Furthermore, MnTMPyP, a manganese-superoxide dismutase mimetic, significantly inhibited IM-induced autophagy and subsequent apoptosis as well as activation of the ERK/Nrf2/HO-1 pathway. These data suggest that autophagy deficiency and subsequent activation of the ERK/Nrf2/HO-1 pathway diminished IM-induced, apoptosis-mediated intestinal epithelial cell damage, and genetic analyses of single nucleotide polymorphisms in autophagy-related genes could predict NSAID-induced intestinal injury.

Metformin increases degradation of phospholamban via autophagy in cardiomyocytes.

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [(35)S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.

Inhibition of phospholamban phosphorylation by O-GlcNAcylation: implications for diabetic cardiomyopathy.

Cardiac-type sarco(endo)plasmic reticulum Ca(2)-ATPase (SERCA2a) plays a major role in cardiac muscle contractility. Phospholamban (PLN) regulates the function of SERCA2a via its Ser(16)-phosphorylation. Since it has been proposed that the Ser/Thr residues on cytoplasmic and nuclear proteins are modified by O-linked N-acetylglucosamine (O-GlcNAc), we examined the effect of O-GlcNAcylation on PLN function in rat adult cardiomyocytes. Studies using enzymatic labeling and co-immunoprecipitation of wild type and a series of mutants of PLN showed that PLN was O-GlcNAcylated and Ser(16) of PLN might be the site for O-GlcNAcylation. In cardiomyocytes treated with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), the O-GlcNAcylation was significantly increased compared to non-treated cells. Simultaneously, Ser(16)-phosphorylation of PLN was reduced. In Chinese hamster ovary cells where PLN cDNA and O-GlcNAc transferase siRNA were co-transfected, the Ser(16)-phosphorylation of PLN was significantly increased compared to controls. The same results were observed in heart homogenates from diabetic rats. In a co-immunoprecipitation of PLN with SERCA2a, the physical interaction between the two proteins was increased in PUGNAc-treated cardiomyocytes. Unlike non-treated cells, the activity of SERCA2a and the profiles of calcium transients in PUGNAc-treated cardiomyocytes were not significantly changed even after treatment with catecholamine. These data suggest that PLN is O-GlcNAcylated to induce the inhibition of its phosphorylation, which correlates to the deterioration of cardiac function. This might define a novel mechanism by which PLN regulation of SERCA2a is altered under conditions where O-GlcNAcylation is increased, such as those occurring in diabetes.

Core fucosylation of E-cadherin enhances cell-cell adhesion in human colon carcinoma WiDr cells.

Alpha1,6-fucosyltransferase (Fut8), an enzyme that catalyzes the introduction of alpha1,6 core fucose to the innermost N-acetylglucosamine residue of the N-glycan, has been implicated in the development, immune system, and tumorigenesis. We found that alpha1,6-fucosyltransferase and E-cadherin expression levels are significantly elevated in primary colorectal cancer samples. Interestingly, low molecular weight population of E-cadherin appeared as well as normal sized E-cadherin in cancer samples. To investigate the correlation between alpha1,6-fucosyltransferase and E-cadherin expression, we introduced alpha1,6-fucosyltransferase in WiDr human colon carcinoma cells. It was revealed that the low molecular weight population of E-cadherin was significantly increased in alpha1,6-fucosyltransferase-transfected WiDr cells in dense culture, which resulted in an enhancement in cell-cell adhesion. The transfection of mutated alpha1,6-fucosyltransferase with no enzymatic activity had no effect on E-cadherin expression, indicating that core fucosylation is involved in the phenomena. In alpha1,6-fucosyltransferase knock down mouse pancreatic acinar cell carcinoma TGP49 cells, the expression of E-cadherin and E-cadherin dependent cell-cell adhesion was decreased. The introduction of alpha1,6-fucosyltransferase into kidney epithelial cells from alpha1,6-fucosyltransferase(-/-) mice restored the expression of E-cadherin and E-cadherin-dependent cell-cell adhesion. Based on the results of lectin blotting, peptide N-glycosidase F treatment, and pulse-chase studies, it was demonstrated that the low molecular weight population of E-cadherin contains peptide N-glycosidase F insensitive sugar chains, and the turnover rate of E-cadherin was reduced in alpha1,6-Fucosyltransferase transfectants. Thus, it was suggested that core fucosylation regulates the processing of oligosaccharides and turnover of E-cadherin. These results suggest a possible role of core fucosylation in the regulation of cell-cell adhesion in cancer.

N-glycan of ErbB family plays a crucial role in dimer formation and tumor promotion.

More and more evidence indicates that N-glycan regulates signal transduction by modulating receptor functions. Previous studies suggested that glycosylation of EGFR is involved in dimerization and endocytosis. We further determined the role of N-glycosylation of ErbB family. A series of human ErbB3 mutants that lack each of the 10 N-glycosylation sites were prepared and transfected to Flp-In-CHO cells for stable expression. A crosslinking study showed that Asn 418 to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin, and the heterodimer formation with ErbB2 was also increased. The N418Q mutant of ErbB3 co-expressed with ErbB2 promoted downstream signaling, anchorage-independent cell growth and the tumor growth in athymic mice. These findings suggest that the specific N-glycan in domain III of ErbB family plays an essential role in regulating receptor dimerization and transforming activity. We assume that the N-glycans affect the conformation of ErbB family, which is crucial for their activity. Together with findings from other laboratories, it is suggested that N-glycosylation controls ErbB signaling by various mechanisms.

The Asn418-linked N-glycan of ErbB3 plays a crucial role in preventing spontaneous heterodimerization and tumor promotion.

ErbB2 and ErbB3, two members of the ErbB family, form a high-affinity heregulin coreceptor that elicits potent mitogenic and transforming signals, and clinical studies indicate that these receptors play an important role in tumor incidence and progression. To determine whether N-glycosylation is involved in the function of ErbB3, a series of human ErbB3 molecules devoid of N-glycans were prepared and transfected to Flp-In-CHO cells for stable expression. A cross-linking study showed that the Asn(418) to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin. The wild-type or N418Q mutant of ErbB3 was next coexpressed with ErbB2 in Flp-In-CHO cells, and the effect of N-glycan on heterodimerization was examined. The N418Q mutant of ErbB3 was autodimerized with ErbB2 without ligand stimulation, and receptor tyrosine phosphorylation and subsequent extracellular signal-regulated kinase (ERK) and Akt phosphorylation were promoted in the absence of heregulin. A cell proliferation assay and a soft agar colony formation assay showed that the N418Q mutant of ErbB3 coexpressed with ErbB2 promoted cell proliferation and colony formation in soft agar in an ERK- and Akt-dependent manner. The mutation also promoted the growth of tumors in athymic mice when injected s.c. These findings suggest that the Asn(418)-linked N-glycan in ErbB3 plays an essential role in regulating receptor heterodimerization with ErbB2 and might have an effect on transforming activity.

Introduction of bisecting GlcNAc in N-glycans of adenylyl cyclase III enhances its activity.

Adenylyl cyclases (ACs) catalyze the synthesis of cAMP in response to extracellular and intracellular signals and are responsible for a wide variety of biological activities including cell growth, differentiation, and metabolism. There are nine, currently known, isoforms of transmembrane ACs, and the primary structure of the catalytic unit and the potential N-glycosylation sites are highly conserved among them. The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) to N-glycans. We have been studying the function of GnT-III on signaling molecules. In this study, we report on the effects of a bisecting GlcNAc on AC signaling. We established GnT-III stable expressing cell lines of Neuro-2a mouse neuroblastoma cells and B16 mouse melanoma cells. Forskolin-induced AC activation and downstream signaling, such as the synthesis of cAMP and the phosphorylation of transcriptional factor CRE-binding protein were upregulated in the GnT-III transfectants compared with mock transfectants or a dominant negative mutant of GnT-III-transfected cells. Since endogenous AC expression levels in Neuro-2a and B16 cells were too low to permit the glycosylation status to be examined, AC type III (ACIII) was overexpressed in a stable expression system using Flp-In-293 cells. The N-glycans of ACIII in the GnT-III transfectants were confirmed to be modified by the introduction of a bisecting GlcNAc, and AC activity was found to be significantly up-regulated in the GnT-III transfectants. Thus, the structure of N-glycans of ACIII regulates its enzymatic activity and downstream signaling.

Acrolein produces nitric oxide through the elevation of intracellular calcium levels to induce apoptosis in human umbilical vein endothelial cells: implications for smoke angiopathy.

Acrolein is a highly electrophilic alpha, beta-unsaturated aldehyde, the levels of which are increased in the blood of smokers. To determine if acrolein is involved in the pathology of smoke angiopathy, the effect of acrolein on human umbilical vein endothelial cells (HUVEC) was examined. Intracellular nitric oxide (NO) levels, determined using diaminofluorescein-2 diacetate (DAF-2 DA), an NO sensitive fluorescent dye, were found to be increased after treatment in HUVEC with 10 microM acrolein. The measurement of nitrite with 2,3-diaminonaphthalene and a Western blot analysis revealed that nitrite and S-nitroso-cysteine levels were increased in a dose-dependent manner, confirming that NO production is increased by acrolein. The increase was not reduced by treatment with 10mM N-acetyl-l-cysteine (NAC), an anti-oxidant, but was reduced with 10 microM of the intracellular calcium chelator, 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester. Acrolein-stimulated NO production was significantly reduced by pretreatment with 1mM N(G)-nitro-l-arginine-methyl ester (L-NAME), an NO synthase inhibitor. The cytotoxicity of acrolein was reduced by pretreatment with 10 microM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO), an intracellular NO scavenger, or 1mM L-NAME, whereas it was not reduced by 10mM NAC, 20 microM Curcumin, another peroxide scavenger, or 100 microM Mn(III)TMPyP, a superoxide dismutase mimic. Nuclear staining and a Western blot analysis using an anti-cleaved caspase 3 antibody revealed that the reduced viability of HUVEC by acrolein was due to apoptosis, which was reversed after pretreatment with 0.1mM carboxy-PTIO or 1mM L-NAME. Thus, acrolein increases intracellular calcium production to induce intracellular NO production by a calcium-dependent NO synthase, possibly eNOS, and the excess and rapid increase in NO might lead to the apoptosis of HUVEC. These data suggest that acrolein might be involved in the pathology of smoke angiopathy through the NO-induced apoptosis of endothelial cells.