Dapagliflozin: A sodium-glucose cotransporter 2 inhibitor, attenuates angiotensin II-induced atrial fibrillation by regulating atrial electrical and structural remodeling.

AIM: Atrial fibrillation (AF), the most common arrhythmia, is characterized by atrial electrical and structural remodeling. Previous studies have found that sodium-glucose cotransporter 2 inhibitor (SGLT2i) can protect myocardium in a glucose independent mechanism. But the role of SGLT2i in regulating AF remains largely unknown. This study, we aimed to investigate the effect of Dapagliflozin (DAPA) in reducing AF susceptibility via inhibiting electrical and structural remodeling. METHOD: The mouse model was established by Angiotensin II (2000 ng/kg/min) infusion for 3 weeks, and an in vitro model was generated by stimulating HL-1 and primary mouse fibroblast with Ang II (1 µM) for 24 h. Programmed electrical stimulation, ECG and whole-cell patch clamp were used to detect DAPA effect on atrial electrical remodeling induced by Ang II. To observe DAPA effect on atrial structural remodeling induced by Ang II, we used echocardiographic, H&E and Masson staining to evaluate atrial dilation. To further explore the protective mechanism of DAPA, we adopt in silico molecular docking approaches to investigate the binding affinity of Ang II and CaMKII at Met-281 site. Western blot was to detect expression level of CaMKII, ox-CaMKII, Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1 and Cx40. RESULTS: Ang II induced AF, atrial dilatation and fibrosis, led to atrial electrical and structural remodeling. However, these effects were markedly abrogated by DAPA treatment, a specific SGLT2i. Our observation of atrial electrical activity in mice revealed that DAPA could rescue the prolonged action potential duration (APD) and the abnormal currents of IK1, Ito and INaL triggered by Ang II infusion. DAPA could reduce the binding affinity of Ang II and CaMKII at Met-281 site, which indicated that DAPA may directly alleviate the activation of CaMKII caused by Ang II. DAPA could reduce the upregulation of ox-CaMKII caused by Ang II infusion in atrial tissues. Moreover, DAPA also ameliorated the aberrant expression levels of electrical activity related proteins (Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1 and Cx40) and fibrosis related signal pathways (TGF-ß1, p-smad/smad) caused by Ang II. Furthermore, we confirmed that DAPA, as well as other SGLT2i (EMPA, CANA), could reverse these abnormalities caused by Ang II incubation in HL-1 cells and primary mouse fibroblasts, respectively. CONCLUSION: Overall, our study identifies DAPA, a widely used SGLT2i, contributes to inhibiting Ang II-induced ox-CaMKII upregulation and electrical and structural remodeling to reduce AF susceptibility, suggesting that DAPA may be a potential therapy of treating AF.

Pinobanksin ameliorated DSS-induced acute colitis mainly through modulation of SLC7A11/glutathione-mediated intestinal epithelial ferroptosis.

Inhibition of ferroptosis in intestinal epithelial cells serves as an attractive target for the development of therapeutic strategies for colitis. Pinobanksin, one of the main flavonoids derived from propolis, possesses significant anti-inflammatory effects and inhibits the cell death of several cell lines. Here, we evaluated whether pinobanksin influenced colitis by modulation of epithelial ferroptosis. Mice treated with 2.5% DSS dissolved in sterile distilled water were established for an acute colitis model. The mitochondrial morphology, colonic iron level, lipid peroxidation products MDA/4-HNE, and lipid reactive oxygen species levels were measured to assess ferroptosis in epithelial cells. RNA-seq and functional analyses were performed to reveal key genes mediating pinobanksin-exerted modulation of ferroptosis. We found that pinobanksin, at different doses, induced significant anti-colitis effects and inhibited the elevated ferroptosis in colonic epithelial cells isolated from DSS-treated mice largely by activating GPX4 (negative regulator of ferroptosis). Furthermore, RNA-seq assays indicated that pinobanksin significantly increased the cystine transporter SLC7A11 in colonic tissues from mice with colitis. Depletion of SLC7A11 largely blocked pinobanksin-induced promotion of cystine uptake/glutathione biosynthesis and suppression of ferroptosis in epithelial cells from mice with colitis or IEC-6 cells pretreated with RSL3. Altogether, pinobanksin alleviated DSS-induced colitis largely by inhibition of ferroptosis in epithelial cells. Activation of SLC7A11 by pinobanksin resulted in the promotion of cystine uptake and enhancement of glutathione biosynthesis. This work will provide novel guidance for the clinical use of pinobanksin to treat colitis through inhibition of epithelial ferroptosis.

Administration of USP7 inhibitor p22077 alleviates Angiotensin II (Ang II)-induced atrial fibrillation in Mice.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is an important contributor to mortality and morbidity. Ubquitin-specific protease 7 (USP7), one of the most abundant ubiquitin-specific proteases (USP), participated in many cellular events, such as cell proliferation, apoptosis, and tumourigenesis. However, its role in AF remains unknown. Here, the mice were treated with Ang II infusion to induce the AF model. Echocardiography was used to measure the atrial diameter. Electrical stimulation was programmed to measure the induction and duration of AF. The changes in atrial remodeling were measured using routine histologic analysis. Here, a significant increase in USP7 expression was observed in Ang II-stimulated atrial cardiomyocytes and atrial tissues, as well as in atrial tissues from patients with AF. The administration of p22077, the inhibitor of USP7, attenuated Ang II-induced inducibility and duration of AF, atrial dilatation, connexin dysfunction, atrial fibrosis, atrial inflammation, and atrial oxidase stress, and then inhibited the progression of AF. Mechanistically, the administration of p22077 alleviated Ang II-induced activation of TGF-ß/Smad2, NF-κB/NLRP3, NADPH oxidases (NOX2 and NOX4) signals, the up-regulation of CX43, ox-CaMKII, CaMKII, Kir2.1, and down-regulation of SERCA2a. Together, this study, for the first time, suggests that USP7 is a critical driver of AF and revealing USP7 may present a new target for atrial fibrillation therapeutic strategies.

Administration of USP7 inhibitor P22077 inhibited cardiac hypertrophy and remodeling in Ang II-induced hypertensive mice.

Hypertension is one of the common causes of pathological cardiac hypertrophy and a major risk for morbidity and mortality of cardiovascular diseases worldwide. Ubiquitin-Specific Protease 7 (USP7), the first identified deubiquitinating enzymes, participated in a variety of biological processes, such as cell proliferation, DNA damage response, tumourigenesis, and apoptosis. However, its role and mechanism in cardiac remodeling remain unclear. Here, our data indicated that USP7 expression was increased during Ang II-induced cardiac hypertrophy and remodeling in mice and humans with heart failure, while the administration of its inhibitor p22077 attenuated cardiac hypertrophy, cardiac fibrosis, inflammation, and oxidase stress. Mechanistically, the administration of p22077 inhibited the multiple signaling pathways, including AKT/ERK, TGF-ß/SMAD2/Collagen I/Collagen III, NF-κB/NLRP3, and NAPDH oxidases (NOX2 and NOX4). Taken together, these findings demonstrate that USP7 may be a new therapeutic target for hypertrophic remodeling and HF.

Cardiac-specific knockdown of Bhlhe40 attenuates angiotensin II (Ang II)-Induced atrial fibrillation in mice.

Atrial fibrosis and atrial inflammation are associated with the pathogenesis of atrial fibrillation (AF). Basic helix-loop-helix family member E40 (Bhlhe40) is an important transcription factor, which is involved in tumors, inflammation, apoptosis, viral infection, and hypoxia. However, its role and molecular mechanism in AF remain unclear. In this study, a mouse model of AF was induced by Ang II infusion. The atrial diameter was evaluated using echocardiography. Induction and duration of AF were measured by programmed electrical stimulation. Atrial structural remodeling was detected using routine histologic examinations. Our results showed that Bhlhe40 was significantly upregulated in angiotensin II (Ang II)-stimulated atrial cardiomyocytes and atrial tissues and in tissues from patients with AF. Cardiac-specific knockdown of Bhlhe40 in mice by a type 9 recombinant adeno-associated virus (rAAV9)-shBhlhe40 significantly ameliorated Ang II-induced atrial dilatation, atrial fibrosis, and atrial inflammation, as well as the inducibility and duration of AF. Mechanistically, cardiac-specific knockdown of Bhlhe40 attenuated Ang II-induced activation of NF-κB/NLRP3, TGF-1ß/Smad2 signals, the increased expression of CX43, and the decreased expression of Kv4.3 in the atria. This is the first study to suggest that Bhlhe40 is a novel regulator of AF progression, and identifying Bhlhe40 may be a new therapeutic target for hypertrophic remodeling and heart failure.

SUMOylation of SYNJ2BP-COX16 promotes breast cancer progression through DRP1-mediated mitochondrial fission.

Treatments targeting oncogenic fusion proteins are notable examples of successful drug development. Abnormal splicing of genes resulting in fusion proteins is a critical driver of various tumors, but the underlying mechanism remains poorly understood. Here, we show that SUMOylation of the fusion protein Synaptojanin 2 binding protein-Cytochrome-c oxidase 16 (SYNJ2BP-COX16) at K107 induces mitochondrial fission in breast cancer and that the K107 site regulates SYNJ2BP-COX16 mitochondrial subcellular localization. Compared with a non-SUMOylated K107R mutant, wild-type SYNJ2BP-COX16 contributed to breast cancer cell proliferation and metastasis in vivo and in vitro by increasing adenosine triphosphate (ATP) production and cytochrome-c oxidase (COX) activity. SUMOylated SYNJ2BP-COX16 recruits dynamin-related protein 1 (DRP1) to the mitochondria to promote ubiquitin-conjugating enzyme 9 (UBC9) binding to DRP1, enhance SUMOylation of DRP1 and phosphorylation of DRP1 at S616, and then induce mitochondrial fission. Moreover, Mdivi-1, an inhibitor of DRP1 phosphorylation, decreased the localization of DRP1 in mitochondria, and prevents SYNJ2BP-COX16 induced mitochondrial fission, cell proliferation and metastasis. Based on these data, SYNJ2BP-COX16 promotes breast cancer progression through the phosphorylation of DRP1 and subsequent induction of mitochondrial fission, indicating that SUMOylation at the K107 residue of SYNJ2BP-COX16 is a novel potential treatment target for breast cancer.

Melatonin inhibits angiotensin II-induced atrial fibrillation through preventing degradation of Ang II Type I Receptor-Associated Protein (ATRAP).

Angiotensin II (Ang II) induced Atrial fibrillation (AF) often accompanied with reduced ATRAP which is a negative modulator of Ang II type 1 receptor (AT1R). Melatonin can protect against AF, but the underlying molecular mechanism remains poorly understood. In this study, Ang II was used to induce AF, and AF inducibility and duration were documented telemetrically. Ang II-infused mice had a higher AF incidence, which was associated with atrial fibrosis, inflammation, and oxidative stress. Melatonin partially inhibited these effects, and enforced expression of siRNA-ATRAP in atria counteracted the beneficial role of melatonin. Specifically, melatonin inhibited expression of Ang II-induced proteasome and immunoproteasome subunits ß2, ß2i, ß5, and ß5i as well as their corresponding trypsin-like and chymotrypsin-like activities and blocked ATRAP degradation. In turn, this inhibited AT1R-mediated NF-κB signaling, transforming growth factor (TGF)-ß1/Smad signaling in the atria, and thereby affected atrial remodeling and AF. Melatonin receptor inhibition by the chemical inhibitor luzindole partially inhibited the inhibitory effects of melatonin on proteasome activity and also Ang II-induced pathological changes in the atria. Overall, our study demonstrates that melatonin protects against Ang II-induced AF by inhibiting proteasome activity and stabilizing ATRAP expression, and these effects are partially dependent on melatonin receptor activation.

Protective Effect of Uric Acid on ox-LDL-Induced HUVECs Injury via Keap1-Nrf2-ARE Pathway.

Uric acid is an effective antioxidant. Oxidized low-density lipoprotein (ox-LDL) is derived from circulating LDL and promotes atherosclerosis. The Keap1-Nrf2-ARE pathway is a key body pathway involved in protection against internal and external oxidative damages. The role of uric acid on vascular endothelial function damaged by ox-LDL, and its effect on the Keap1-Nrf2-ARE pathway has not been fully explored. HUVECs were treated with different concentrations of uric acid and ox-LDL to explore the effect of uric acid in vitro. Cell phenotype was determined by cytometry and Western blot. Nuclear translocation of Nrf2 was determined by immunofluorescence. Coimmunoprecipitation was used to determine the level of Nrf2 ubiquitination. A microfluidic device was used to mimic the vascular environment in the body, and the level of mRNA levels of inflammatory factors was determined by RT-PCR. The findings of this study show that suitable uric acid can significantly reduce endothelial damage caused by ox-LDL, such as oxidative stress, inflammation, and increased adhesion. In addition, uric acid reduced Nrf2 ubiquitination and increased nuclear translocation of Nrf2 protein, thus activating the Keap1-Nrf2-ARE pathway and playing a protective role. Interestingly, the effects of UA were significantly inhibited by administration of Brusatol, an inhibitor of Nrf2. In summary, suitable concentrations of uric acid can alleviate the oxidative stress level of endothelial cells through Nrf2 nuclear translocation and further protect cells from damage.

Rutaecarpine Inhibits Doxorubicin-Induced Oxidative Stress and Apoptosis by Activating AKT Signaling Pathway.

Patients with cancer who receive doxorubicin (DOX) treatment can experience cardiac dysfunction, which can finally develop into heart failure. Oxidative stress is considered the most important mechanism for DOX-mediated cardiotoxicity. Rutaecarpine (Rut), a quinazolinocarboline alkaloid extracted from Evodia rutaecarpa was shown to have a protective effect on cardiac disease. The purpose of this study is to investigate the role of Rut in DOX-induced cardiotoxicity and explore the underlying mechanism. Intravenous injection of DOX (5 mg/kg, once a week) in mice for 4 weeks was used to establish the cardiotoxic model. Echocardiography and pathological staining analysis were used to detect the changes in structure and function in the heart. Western blot and real-time PCR analysis were used to detect the molecular changes. In this study, we found that DOX time-dependently decreased cardiac function with few systemic side effects. Rut inhibited DOX-induced cardiac fibrosis, reduction in heart size, and decrease in heart function. DOX-induced reduction in superoxide dismutase (SOD) and glutathione (GSH), enhancement of malondialdehyde (MDA) was inhibited by Rut administration. Meanwhile, Rut inhibited DOX-induced apoptosis in the heart. Importantly, we further found that Rut activated AKT or nuclear factor erythroid 2-related factor 2 (Nrf-2) which further upregulated the antioxidant enzymes such as heme oxygenase-1 (HO-1) and GSH cysteine ligase modulatory subunit (GCLM) expression. AKT inhibitor (AKTi) partially inhibited Nrf-2, HO-1, and GCLM expression and abolished the protective role of Rut in DOX-induced cardiotoxicity. In conclusion, this study identified Rut as a potential therapeutic agent for treating DOX-induced cardiotoxicity by activating AKT.

Selective Inhibition of the Immunoproteasome ß5i Prevents PTEN Degradation and Attenuates Cardiac Hypertrophy.

Cardiac hypertrophy without appropriate treatment eventually progresses to heart failure. Our recent data demonstrated that the immunoproteasome subunit ß5i promotes cardiac hypertrophy. However, whether ß5i is a promising therapeutic target for treating hypertrophic remodeling remains unknown. Here, we investigated the effects of PR-957, a ß5i-specific inhibitor, on angiotensin II (Ang II)-induced hypertrophic remodeling in the murine heart. The infusion of Ang II increased immunoproteasome chymotrypsin-like activity and ß5i catalytic subunit expression in the heart, whereas PR-957 treatment fully blocked the enhanced immunoproteasome activity caused by Ang II. Moreover, the administration of PR-957 significantly suppressed Ang II-induced cardiac hypertrophy, fibrosis, and inflammation. Mechanistically, PR-957 treatment inhibited phosphatase and tensin homolog on chromosome ten (PTEN) degradation, thereby inhibiting multiple signals including AKT/mTOR, ERK1/2, transforming growth factor-ß, and IKB/NF-kB. Furthermore, PTEN blocking by its specific inhibitor VO-OHpic markedly attenuated the inhibitory effect of PR-957 on Ang II-induced cardiac hypertrophy in mice. We conclude that PR-957 blocks PTEN degradation and activates its downstream mediators, thereby attenuating Ang II-induced cardiac hypertrophy. These findings highlight that PR-957 may be a potential therapeutic agent for Ang II-induced hypertrophic remodeling.

The deubiquitinase UCHL1 regulates cardiac hypertrophy by stabilizing epidermal growth factor receptor.

Pathological cardiac hypertrophy leads to heart failure (HF). The ubiquitin-proteasome system (UPS) plays a key role in maintaining protein homeostasis and cardiac function. However, research on the role of deubiquitinating enzymes (DUBs) in cardiac function is limited. Here, we observed that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) was significantly up-regulated in agonist-stimulated primary cardiomyocytes and in hypertrophic and failing hearts. Knockdown of UCHL1 in cardiomyocytes and mouse hearts significantly ameliorated cardiac hypertrophy induced by agonist or pressure overload. Conversely, overexpression of UCHL1 had the opposite effect in cardiomyocytes and rAAV9-UCHL1-treated mice. Mechanistically, UCHL1 bound, deubiquitinated, and stabilized epidermal growth factor receptor (EGFR) and activated its downstream mediators. Systemic administration of the UCHL1 inhibitor LDN-57444 significantly reversed cardiac hypertrophy and remodeling. These findings suggest that UCHL1 positively regulates cardiac hypertrophy by stabilizing EGFR and identify UCHL1 as a target for hypertrophic therapy.

Inhibition of UCHL1 by LDN-57444 attenuates Ang II-Induced atrial fibrillation in mice.

Atrial fibrillation (AF) is the most common human arrhythmia in clinical practice and may be promoted by atrial inflammation and fibrosis. Ubiquitination is an important posttranslational modification process that is reversed by deubiquitinating enzymes (DUBs). DUBs play critical roles in modulating the degradation, activity, trafficking, and recycling of substrates. However, less research has focused on the role of DUBs in AF. Here, we investigated the effect of ubiquitin C-terminal hydrolase 1 (UCHL1), an important DUB, on the development of AF induced by angiotensin II (Ang II). Male wild-type mice were treated with the UCHL1 inhibitor LDN57444 (LDN) at a dose of 40 µg/kg and infused with Ang II (2000 ng/kg/min) for 3 weeks. Our results showed that Ang II-infused wild-type (WT) mice had higher systolic blood pressure and an increased incidence and duration of AF. Conversely, this effect was attenuated in LDN-treated mice. Moreover, the administration of LDN significantly reduced Ang II-induced left atrial dilation, fibrosis, inflammatory cell infiltration, and reactive oxygen species (ROS) production. Mechanistically, LDN treatment inhibited the activation of multiple signaling pathways (the AKT, ERK1/2, HIF-1α, and TGF-ß/smad2/3 pathways) and the expression of CX43 protein in atrial tissues compared with that in vehicle-treated control mice. Overall, our study identified UCHL1 as a novel regulator that contributes to Ang II-induced AF and suggests that the administration of LDN may represent a potential therapeutic approach for treating hypertensive AF.

The immunoproteasome catalytic ß5i subunit regulates cardiac hypertrophy by targeting the autophagy protein ATG5 for degradation.

Pathological cardiac hypertrophy eventually leads to heart failure without adequate treatment. The immunoproteasome is an inducible form of the proteasome that is intimately involved in inflammatory diseases. Here, we found that the expression and activity of immunoproteasome catalytic subunit ß5i were significantly up-regulated in angiotensin II (Ang II)-treated cardiomyocytes and in the hypertrophic hearts. Knockout of ß5i in cardiomyocytes and mice markedly attenuated the hypertrophic response, and this effect was aggravated by ß5i overexpression in cardiomyocytes and transgenic mice. Mechanistically, ß5i interacted with and promoted ATG5 degradation thereby leading to inhibition of autophagy and cardiac hypertrophy. Further, knockdown of ATG5 or inhibition of autophagy reversed the ß5i knockout-mediated reduction of cardiomyocyte hypertrophy induced by Ang II or pressure overload. Together, this study identifies a novel role for ß5i in the regulation of cardiac hypertrophy. The inhibition of ß5i activity may provide a new therapeutic approach for hypertrophic diseases.

Resveratrol as a new inhibitor of immunoproteasome prevents PTEN degradation and attenuates cardiac hypertrophy after pressure overload.

Sustained cardiac hypertrophy is a major cause of heart failure (HF) and death. Recent studies have demonstrated that resveratrol (RES) exerts a protective role in hypertrophic diseases. However, the molecular mechanisms involved are not fully elucidated. In this study, cardiac hypertrophic remodeling in mice were established by pressure overload induced by transverse aortic constriction (TAC). Cardiac function was evaluated by echocardiography and invasive pressure-volume analysis. Cardiomyocyte size was detected by wheat germ agglutinin staining. The protein and gene expressions of signaling mediators and hypertrophic markers were examined. Our results showed that administration of RES significantly suppressed pressure overload-induced cardiac hypertrophy, fibrosis and apoptosis and improved in vivo heart function in mice. RES also reversed pre-established hypertrophy and restoring contractile dysfunction induced by chronic pressure overload. Moreover, RES treatment blocked TAC-induced increase of immunoproteasome activity and catalytic subunit expression (ß1i, ß2i and ß5i), which inhibited PTEN degradation thereby leading to inactivation of AKT/mTOR and activation of AMPK signals. Further, blocking PTEN by the specific inhibitor VO-Ohpic significantly attenuated RES inhibitory effect on cardiomyocyte hypertrophy in vivo and in vitro. Taken together, our data suggest that RES is a novel inhibitor of immunoproteasome activity, and may represent a promising therapeutic agent for the treatment of hypertrophic diseases.

Administration of ubiquitin-activating enzyme UBA1 inhibitor PYR-41 attenuates angiotensin II-induced cardiac remodeling in mice.

Pathological cardiac hypertrophy is the main risk factor for heart diseases. The ubiquitin-proteasome system (UPS) is the major intracellular protein degradation system involved in the development of cardiac hypertrophic remodeling. Ubiquitin-activating enzyme E1, a key component of the UPS, catalyzes the first step in ubiquitin conjugation to mark cellular proteins for degradation via proteasome. However, the functional role of E1 (UBA1) in regulation of hypertrophic remodeling in angiotensin II (Ang II)-infused mice remains unknown. In this study, male wild-type mice were treated with UBA1 inhibitor PYR-41 at two doses of 5 and 10 mg and infused with Ang II (1000 ng/kg/min) for 14 days. Systolic blood pressure was detected by using tail-cuff system. Cardiac function was assessed by echocardiography. Hypertrophic remodeling was analyzed examined by histological examinations. The expressions of genes and proteins were detected by quantitative real-time PCR and immunoblotting analysis. After 14 days, Ang II infusion significantly increased UBA1 expression at both mRNA and protein levels in the hearts. Furthermore, Ang II-infused mice showed a significant increase in systolic blood pressure compensatory cardiac function, hypertrophy, interstitial fibrosis, inflammation and oxidative stress compared with saline-treated controls, whereas these effects were dose-dependently attenuated in PYR-41-treated mice. These beneficial actions were associated mainly with inhibition of PTEN degradation and multiple downstream mediators (AKT, ERK1/2, STAT3, TGF-ß/Smad2/3 and NF-kB(p65)). In conclusion, these results indicate that inhibition of UBA1 suppresses Ang II-induced hypertrophic remodeling, and suggest that administration of low dose PYR-41 may be a new potential therapeutic approach for treating hypertensive heart diseases.

Knockout of immunoproteasome subunit ß2i ameliorates cardiac fibrosis and inflammation in DOCA/Salt hypertensive mice.

The immunoproteasome is a multicatalytic protease complex in all eukaryotic cells, which plays a key role in regulating essential cellular processes. However, the role of immunoproteasome subunit ß2i in regulation of cardiac fibrosis and inflammation in deoxycorticosterone-acetate (DOCA)/salt mice remains unknown. Wild-type (WT) and ß2i knockout (KO) mice were subjected to uninephrectomy and DOCA/salt treatment for 21 days. Blood pressure was measured by the tail-cuff system. Cardiac function and remodeling were examined by echocardiography, hematoxylin-eosin (H&E) and Masson's trichrome staining. The gene and protein expressions were detected by quantitative real-time PCR, and Western blot analysis. After 21 days, DOCA/salt treatment significantly up-regulated the expression of ß2i mRNA and protein in the hearts. Moreover, systolic blood pressure and heart weight/body weight (HW/BW) ratio were significantly higher in DOCA/salt mice than in sham groups, and these effects were markedly reversed in ß2i knockout mice. Importantly, DOCA/salt-induced cardiac fibrosis, inflammation and the expression of collagen I, collagen III, α-SMA, IL-1ß, IL-6 and TNF-α in the wild-type hearts, which were markedly attenuated by ß2i knockout. These beneficial effects were due, at least in part, to the inhibition of IκBα/NF-κB and TGF-ß1/Smad2/3 signaling pathways. Collectively, these findings indicate that knockout of ß2i ameliorates DOCA/salt-induced cardiac fibrosis and inflammation, and may be a novel potential therapeutic target for hypertensive heart diseases.

Protection against doxorubicin-induced myocardial dysfunction in mice by cardiac-specific expression of carboxyl terminus of hsp70-interacting protein.

Carboxyl terminus of Hsp70-interacting protein (CHIP) is a critical ubiquitin ligase/cochaperone to reduce cardiac oxidative stress, inflammation, cardiomyocyte apoptosis and autophage etc. However, it is unclear whether overexpression of CHIP in the heart would exert protective effects against DOX-induced cardiomyopathy. Cardiac-specific CHIP transgenic (CHIP-TG) mice and the wild-type (WT) littermates were treated with DOX or saline. DOX-induced cardiac atrophy, dysfunction, inflammation, oxidative stress and cardiomyocyte apoptosis were significantly attenuated in CHIP-TG mice. CHIP-TG mice also showed higher survival rate than that of WT mice (40% versus 10%) after 10-day administration of DOX. In contrast, knockdown of CHIP by siRNA in vitro further enhanced DOX-induced cardiotoxic effects. Global gene microarray assay revealed that after DOX-treatment, differentially expressed genes between WT and CHIP-TG mice were mainly involved in apoptosis, atrophy, immune/inflammation and oxidative stress. Mechanistically, CHIP directly promotes ubiquitin-mediated degradation of p53 and SHP-1, which results in activation of ERK1/2 and STAT3 pathways thereby ameliorating DOX-induced cardiac toxicity.

SUMOylation of GPS2 protein regulates its transcription-suppressing function.

G-protein pathway suppressor 2 (GPS2) is a human suppressor of G protein-activated mitogen-activated protein kinase signaling. It is involved in many physiological processes, including DNA repair, cell proliferation, apoptosis, and brain development. In this study, we show that GPS2 can be modified by the small ubiquitin-like modifier (SUMO) SUMO-1 but not SUMO-2 or -3. Two SUMOylation sites (K45 and K71) are identified in the N-terminal coiled-coil domain of GPS2. Substitution of K45 with arginine reduces SUMOylation, whereas substitution of K71 or both K45 and K71 with arginine abolishes SUMOylation, with more of the double mutant GPS2 appearing in the cytosol than in the nucleus compared with wild type and the two-single-mutant GPS2. SUMOylation stabilizes GPS2 protein by promoting its interaction with TBL1 and reducing its ubiquitination. SUMOylation also enhances the ability of GPS2 to suppress transcription and promotes its ability to inhibit estrogen receptor α-mediated transcription by increasing its association with SMRT, as demonstrated in MCF-7 and T47D cells. Moreover, SUMOylation of GPS2 also represses the proliferation of MCF-7 and T47D cells. These findings suggest that posttranslational modification of GPS2 by SUMOylation may serve as a key factor that regulates the function of GPS2 in vivo.

Induction of the CLOCK gene by E2-ERα signaling promotes the proliferation of breast cancer cells.

Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.