Knockdown Of Uncoupling Protein 2 Research Articles

ACOT1 deficiency attenuates high-fat diet-induced fat mass gain by increasing energy expenditure.

Acyl-CoA thioesterase 1 (ACOT1) catalyzes the hydrolysis of long-chain acyl-CoAs to free fatty acids and CoA and is typically upregulated in obesity. Whether targeting ACOT1 in the setting of high-fat diet-induced (HFD-induced) obesity would be metabolically beneficial is not known. Here we report that male and female ACOT1KO mice are partially protected from HFD-induced obesity, an effect associated with increased energy expenditure without alterations in physical activity or food intake. In males, ACOT1 deficiency increased mitochondrial uncoupling protein-2 (UCP2) protein abundance while reducing 4-hydroxynonenal, a marker of oxidative stress, in white adipose tissue and liver of HFD-fed mice. Moreover, concurrent knockdown (KD) of UCP2 with ACOT1 in hepatocytes prevented increases in oxygen consumption observed with ACOT1 KD during high lipid loading, suggesting that UCP2-induced uncoupling may increase energy expenditure to attenuate weight gain. Together, these data indicate that targeting ACOT1 may be effective for obesity prevention during caloric excess by increasing energy expenditure.

Mitochondria of lung venular capillaries mediate lung-liver crosstalk in pneumonia.

Failure of the lung's endothelial barrier underlies lung injury, which causes the high mortality Acute Respiratory Distress Syndrome (ARDS). Multiple organ failure predisposes to the mortality, but mechanisms are poorly understood. Here, we show that mitochondrial Uncoupling Protein 2 (UCP2), a component of the mitochondrial inner membrane, plays a role in the barrier failure. Subsequent lung-liver crosstalk mediated by neutrophil activation causes liver congestion. We intranasally instilled lipopolysaccharide (LPS). Then, we viewed the lung endothelium by real-time confocal imaging of the isolated, blood-perfused mouse lung. LPS caused alveolar-capillary transfer of reactive oxygen species and mitochondrial depolarization in lung venular capillaries. The mitochondrial depolarization was inhibited by transfection of alveolar Catalase and vascular knockdown of UCP2. LPS instillation caused lung injury as indicated by increases in bronchoalveolar lavage (BAL) protein content and extra-vascular lung water. LPS or Pseudomonas aeruginosa instillation also caused liver congestion, quantified by liver hemoglobin and plasma aspartate aminotransferase (AST) increases. Genetic inhibition of vascular UCP2 prevented both lung injury and liver congestion. Antibody-mediated neutrophil depletion blocked the liver responses, but not lung injury. Knockdown of lung vascular UCP2 mitigated P. aeruginosa-induced mortality. Together, these data suggest a mechanism in which bacterial pneumonia induces oxidative signaling to lung venular capillaries, known sites of inflammatory signaling in the lung microvasculature, depolarizing venular mitochondria. Successive activation of neutrophils induces liver congestion. We conclude, oxidant-induced UCP2 expression in lung venular capillaries causes a mechanistic sequence leading to liver congestion and mortality. Lung vascular UCP2 may present a therapeutic target in ARDS.

Activation of UCP2 by anethole trithione suppresses neuroinflammation after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating disease, in which neuroinflammation substantially contributes to brain injury. Uncoupling protein 2 (UCP2) is a member of the mitochondrial anion carrier family, which uncouples oxidative phosphorylation from ATP synthesis by facilitating proton leak across the mitochondrial inner membrane. UCP2 has been reported to modulate inflammation. In this study we investigated whether and how UCP2 modulated neuroinflammation through microglia/macrophages following ICH in vitro and in vivo. We used an in vitro neuroinflammation model in murine BV2 microglia to mimic microglial activation following ICH. ICH in vivo model was established in mice through collagenase infusion into the left striatum. ICH mice were treated with anetholetrithione (ADT, 50 mg· kg-1 ·d-1, ip) or the classical protonophoric uncoupler FCCP (injected into hemorrhagic striatum). We showed that the expression and mitochondrial location of microglial UCP2 were not changed in both in vitro and in vivo ICH models. Knockdown of UCP2 exacerbated neuroinflammation in BV2 microglia and mouse ICH models, suggesting that endogenous UCP2 inhibited neuroinflammation and therefore played a protective role following ICH. ADT enhanced mitochondrial ROS production thus inducing mitochondrial uncoupling and activating UCP2 in microglia. ADT robustly suppressed neuroinflammation, attenuated brain edema and improved neurological deficits following ICH, and these effects were countered by striatal knockdown of UCP2. ADT enhanced AMP-activated protein kinase (AMPK) activation in the hemorrhagic brain, which was abrogated by striatal knockdown of UCP2. Moreover, striatal knockdown of AMPK abolished the suppression of neuroinflammation by ADT following ICH. On the other hand, FCCP-induced mitochondrial uncoupling was independent of UCP2 in microglia; and striatal knockdown of UCP2 did not abrogate the suppression of neuroinflammation by FCCP in ICH mice. In conclusion, the uncoupling activity is essential for suppression of neuroinflammation by UCP2. We prove for the first time the concept that activators of endogenous UCP2 such as anetholetrithione are a new class of uncouplers with translational significance.

Antileishmanial effect of the natural immunomodulator genipin through suppression of host negative regulatory protein UCP2.

To evaluate the antileishmanial efficacy of genipin, which specifically inhibits uncoupling protein 2 (UCP2) that is induced in leishmaniasis to neutralize reactive oxygen species (ROS). The effect of genipin was assessed against intracellular parasites in cultured macrophages and in suppressing spleen and liver parasite burdens in a BALB/c mouse model of visceral leishmaniasis by microscopic evaluation of intracellular amastigotes stained with Giemsa. ROS and mitochondrial membrane potential were measured by H2DCFDA- and JC-1-based fluorometric analysis. ELISA was performed for various Th1 and Th2 cytokines in both in vitro and in vivo infected conditions to evaluate the type of immunological responses. The role of UCP2 was assessed by lipofectamine-mediated transfection and overexpression in macrophages and short hairpin RNA-mediated knockdown of UCP2 in infected animals. Genipin reduced the infection-induced UCP2 levels in macrophages, with optimum effect at 100 μM. Genipin reversed parasite-induced ROS suppression and mitochondrial membrane potential disruption. It has no inhibitory effect on promastigote or axenic amastigote forms, but markedly suppressed amastigote multiplication within macrophages, which was reversed by the ROS scavenger N-acetyl cysteine. Genipin administration (30 mg/kg/day) in infected mice showed significant suppression of liver and spleen parasite burdens with an enhanced host-favourable cytokine balance in a ROS-p38 mitogen-activated protein kinase-dependent manner. Co-treatment with genipin plus a sublethal dose of sodium antimony gluconate (SAG50) showed almost a curative reduction in spleen and liver parasite burden. These results suggest the effectiveness of genipin as a synergistic agent for the front-line antileishmanial drug SAG in circumventing the resistance and toxicity problems associated with its high curative dose.

The Warburg Effect Promotes Mitochondrial Injury Regulated by Uncoupling Protein-2 in Septic Acute Kidney Injury.

Evidence implying that metabolism reprogramming plays an important role in the regulation of sepsis is increasing; however, whether it has a similar role in septic organ dysfunction remains unclear. Here, we provide evidence to support a new role of uncoupling protein-2 (UCP2)-regulated Warburg effect, i.e., aerobic glycolysis, in promoting mitochondrial injury in the kidney. To imitate sepsis condition, male C57BL/6 mice were operated by the cecal ligation puncture in vivo, whereas a normal human kidney cell line (HK-2) was treated with lipopolysaccharide in vitro. UCP2 small interfering RNA pretreatment was performed to knock down UCP2 expression in vitro. The glycolysis metabolite was detected by liquid chromatography/tandem mass spectrometry in vivo and detected by commercial kits in vitro. Oxidative phosphorylation level and glycolysis level were monitored by measuring the oxygen consumption rate (indicative of respiration) and extracellular acidification rate (indicative of glycolysis) in vitro. Exogenous lactate was supplied to stimulate HK-2 cells and indicators of mitochondrial dysfunction were also assessed. Aerobic glycolysis is enhanced in septic tubular epithelial cells, and the glycolysis inhibitor 2-deoxyglucose can partially restore mitochondrial membrane potential and decrease the reactive oxygen species production. With the knockdown of UCP2, the aerobic glycolysis level upregulates, and mitochondrial injury increases. These results provide insights on a new mechanism of metabolic regulation of mitochondrial injury and the importance of targeting aerobic glycolysis for the treatment of septic acute kidney injury.

Targeting UCP2 Suppresses the FAK Signaling and Progression of Human Head and Neck Cancer Cells

Background: New adjuvant therapies for human head and neck (H&N) cancer to improve the quality of life of the patients are in great demand. Our early studies have demonstrated that uncoupling protein 2 (UCP2) is upregulated in the tumor tissues of H&N cancer compared to the adjacent normal tissues; however, the role of UCP2 in H&N cancer has not been studied. Objective: In this manuscript, we aim to examine whether UCP2 contributes to H&N cancer progression in vitro. Methods: We generated UCP2 stable knockdown H&N cancer cells and detected the effects of UCP2 inhibition on cell proliferation, migration, invasion, 3D spheroid formation, and the sensitivity to a chemodrug treatment. Results: Knockdown of UCP2 suppressed the progression of H&N cancer in vitro, which might be mediated via the following mechanism: 1) increased the G1 phase whereas decreased the S phase of the cell cycle, which could be mediated by suppression of the G1/S regulators including CDK4/6 and cyclin D1. 2) Decreased mitochondrial oxygen consumption, ATP production, and lactate formation, which is consistent with the downregulation of c-Myc. 3) FAK may serve as the upstream signaling molecule, and its action was mediated by Akt and ERK. Conclusions: Our studies first demonstrate that targeting UCP2 may suppress H&N cancer progression in vitro.

Uncoupling protein 2 is upregulated in melanoma cells and contributes to the activation of Akt/mTOR and ERK signaling

The aim of the present study was to characterize the expression of uncoupling protein2 (UCP2) in melanoma and to study the potential mechanisms underlying the involvement of UCP2 in melanomagenesis using human melanoma cell lines. The expression of UCP2 was evaluated in specimens from normal control subjects, patients with compound nevus, and patients with cutaneous and mucosal melanoma. Stable knockdown of UCP2 was achieved in human melanoma cell lines, which were used to examine whether UCP2 knockdown affects the mitochondrial membrane potential and intracellular levels of ATP, reactive oxygen species and lactate. Cell proliferation, invasion, spheroid formation and cisplatin sensitivity were also evaluated in the UCP2 knockdown cells. Finally, the effects of UCP2 knockdown on the Akt/mammalian target of rapamycin (mTOR) and extracellular signal‑regulated kinase (ERK) pathways, which are important oncogenic pathways during melanomagenesis, were evaluated. Relatively high expression of UCP2 was detected in human melanoma specimens, which was correlated with Clark level and Breslow thickness. Knockdown of UCP2 suppressed cell proliferation, invasion and spheroid formation, and increased the sensitivity of melanoma cells to cisplatin. Furthermore, the UCP2 knockdown cells exhibited inhibition of Akt/mTOR signaling and ERK activation. Therefore, human melanoma tissues exhibit relatively high UCP2 expression, which may be implicated in the mechanisms underlying tumor progression. The potential role of UCP2 in melanomagenesis may involve enhancing the Akt/mTOR and mitogen‑activated protein kinase/ERK pathways.

UCP2 silencing aggravates mitochondrial dysfunction in astrocytes under septic conditions

Uncoupling protein 2 (UCP2) plays a positive role in sepsis. However, the role of UCP2 in experimental sepsis in astrocytes remains unknown. The present study was designed to determine whether UCP2 has a protective effect in an experimental sepsis model in astrocytes asnd to clarify the mechanisms responsible for its neuroprotective effects after sepsis. An experimental astrocyte model mimicking sepsis-induced brain injury was established using lipopolysaccharide (LPS) and interferon (IFN)-γ. Additionally, UCP2 knockdown in astrocytes was achieved by adenovirus transfection. Tumor necrosis factor (TNF)-α and interleukin (IL)-1β activity, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels were assessed. The mitochondrial ultrastructure was evaluated, and the expression of UCP2 was determined by western blotting. LPS with IFN-γ co-stimulation increased the mRNA and protein expression levels of UCP2 in astrocytes, damaged the mitochondrial structure, and accelerated the release of TNF-α and IL-1β, resulting in a decrease in the MMP, and the excessive generation of ROS. Moreover, sepsis also caused a reduction in ATP production. The knockdown of UCP2 exacerbated astrocyte injury and mitochondrial impairment. In conclusion, both the function and morphology of mitochondria were damaged in an experimental model of sepsis in astrocytes, and knockdown of UCP2 using shRNA exacerbated this impairment, suggesting that UCP2 has a positive effect on astrocytes as determined in an experimental sepsis model.

Enhancing Retinal Endothelial Glycolysis by Inhibiting UCP2 Promotes Physiologic Retinal Vascular Development in a Model of Retinopathy of Prematurity.

PurposeWe address the hypothesis that uncoupling protein 2 (UCP2), a cellular glucose regulator, delays physiologic retinal vascular development (PRVD) by interfering with glucose uptake through glucose transporter 1 (Glut1).MethodsIn the rat 50/10 oxygen-induced retinopathy (OIR) model, retinal Glut1 and UCP2 were measured and compared to room air (RA)-raised pups at postnatal day 14 (p14). Pups in OIR and RA received intraperitoneal genipin, an UCP2 inhibitor, or control every other day from p3 until p13. Analyses at p14 included avascular/total retinal area (AVA), Western blots of retinal UCP2 and Glut1, and immunostaining of Glut1 in retinal cryosections. Intravitreal neovascular/total retinal area (IVNV) was analyzed at p18, and electroretinograms were performed at p26. Glut1 and phosphorylated VEGFR2 (p-VEGFR2), glucose uptake, adenosine triphosphate (ATP) production, and cell proliferation were measured in human retinal microvascular endothelial cells (hRMVECs) pretreated with genipin or transfected with UCP2siRNA, Glut1siRNA, or control siRNA when incubated with VEGF or PBS.ResultsAt p14, OIR pups had increased AVA with decreased Glut1 and increased UCP2 in the retina compared to RA retinas. Intraperitoneal genipin increased retinal Glut1 and reduced AVA. Compared to control, treatment with genipin or knockdown of UCP2 significantly increased Glut1, glucose uptake, ATP production, VEGF-induced p-VEGFR2 and cell proliferation in hRMVECs. Knockdown of Glut1 inhibited VEGF-induced p-VEGFR2. Genipin-treated OIR pups with decreased AVA at p14 had reduced IVNV at p18 and increased amplitudes in a- and b-waves at p26.ConclusionsExtending PRVD by increasing retinal endothelial glucose uptake may represent a strategy to prevent severe retinopathy of prematurity and vision loss.

Uncoupling Protein 2 Drives Myocardial Dysfunction in Murine Models of Septic Shock.

Cardiac dysfunction is a major component of sepsis-induced multiorgan failure in critical care units. Uncoupling protein 2 (UCP2) involves immune response, regulation of oxidative stress, and maintenance of mitochondrial membrane potential as well as energy production. However, whether and how UCP2 plays roles in the development of septic cardiac dysfunction are largely unknown. Here, intraperitoneal injection of LPS significantly activated UCP2 expression accompanied by a significant decrease of cardiac function and caused a significantly lower survival rate in mice. Of note, knockdown of UCP2 through a cardiotropic adenoassociated viral vector carrying a short hairpin RNA (shRNA) specifically targeting the UCP2 evoked resistance to LPS-triggered septic cardiac dysfunction and lethality in vivo. Moreover, UCP2 deficiency ameliorated the reduced levels of intracellular ATP in the LPS-challenged heart tissues and preserved mitochondrial membrane potential loss in primary adult mouse cardiomyocytes in LPS-challenged animals. Mechanistically, we confirmed that the inhibition of UCP2 promoted autophagy in response to LPS, as shown by an increase in LC3II and a decrease in p62. At last, the autophagy inhibitor 3-MA abolished UCP2 knockdown-afforded cardioprotective effects. Those results indicate that UCP2 drives septic cardiac dysfunction and that the targeted induction of UCP2-mediated autophagy may have important therapeutic potential.

A Chinese 4-herb formula, Yiqi-Huoxue granule, alleviates H2O2-induced apoptosis by upregulating uncoupling protein 2 in H9c2 cells

BackgroundAlthough the protective effects of Yiqi-Huoxue granule (YQHX), a Chinese 4-herb formula, on patients with ischemic heart diseases are related to the attenuation of oxidative stress injury, the mechanism(s) underlying these actions remains poorly understood. PurposeOur aim was to investigate the potential protective effects of YQHX treatment against oxidative stress induced by hydrogen peroxide (H2O2) in rat H9c2 cells. MethodsH9c2 cells were treated with YQHX for 16 h before exposed to 200 μM H2O2 for 6 h. The apoptosis induced by H2O2 was measured using hoechst 33,342 staining and Annexin-V FITC/PI assay. The expression of uncoupling protein 2 (UCP2), Bcl-2, Bax, and caspase-3 were observed using western blot. The effects of UCP2 knockdown on cell apoptosis and intracellular ROS production were also investigated. ResultsH2O2 exposure led to significant activation of oxidative stress followed by increased apoptosis and ROS production, as well as decreased UCP2 expression in H9c2 cells. YQHX treatment at the concentration of 0.75 and 1.5 mg/ml remarkably reduced the expression of Bax and caspase-3, whereas increased the protein expression of Bcl-2 and UCP2. These changes were attenuated by transgenic knockdown of UCP2 with Lenti-shUCP2 vector. ConclusionsTaken together, our study demonstrated that YQHX attenuates H2O2-induced apoptosis by upregulating UCP2 expression in H9c2 Cells, suggesting that YQHX is a promising therapeutic approach for the treatment of I/R injury-mediated apoptosis.

Targeted upregulation of uncoupling protein 2 within the basal ganglia output structure ameliorates dyskinesia after severe liver failure

Impaired motor function, due to the dysfunction of the basal ganglia, is the most common syndrome of hepatic encephalopathy (HE), and its etiology remains poorly understood. Neural oxidative stress is shown to be the major cellular defects contributing to HE pathogenesis. Mitochondrial uncoupling protein 2 (UCP2) has been implicated in neuroprotection in several neurological disorders. We explored the neuroprotective role of UCP2 within the substantia nigra pars reticulate (SNr), the output structure of the basal ganglia, in HE. The toxin thioacetamide (TAA) induced HE mice showed hypolocomotion, which was associated with decreased ATP levels and loss of antioxidant substances SOD and GSH within the SNr. Stable overexpression of UCP2 via AAV-UCP2 under the control of the UCP2 promoter in bilateral SNr preserved local ATP level, increased antioxidant substances, and ameliorated locomotion defects after severe liver failure. Contrary to UCP2 overexpression, targeted knockdown of UCP2 within bilateral SNr via AAV-UCP2 shRNA exacerbated the impaired mitochondrial dysfunction and hypokinesia in HE mice. The modulatory effects of UCP2 was due to mediation of K+-Cl− cotransporter-2 (KCC2) expression on GABAergic neurons of SNr. Taken together, our results demonstrate that UCP2 exerts a neural protective role at the subcortical level by increasing the resistance of neurons to oxidative stress, which may offer a novel therapeutic target for the treatment of motor dysfunction diseases.

Bile acid and cigarette smoke enhance the aggressive phenotype of esophageal adenocarcinoma cells by downregulation of the mitochondrial uncoupling protein-2.

Limited information is available regarding mechanisms that link the known carcinogenic risk factors of gastro-esophageal reflux and cigarette smoking to metabolic alterations in esophageal adenocarcinoma (EAC). In the present study, we utilized a novel in-vitro model to examine whether bile acid and cigarette smoke increase the aggressiveness of EAC and whether these changes are associated with metabolic changes.EAC cells (EACC) were exposed to 10 μg/ml cigarette smoke condensate (CSC) and/or 100 μM of the oncogenic bile acid, deoxycholic acid (DCA), for 5 days. These exposure conditions were chosen given their lack of effect on proliferation or viability. DCA and CSC increased invasion, migration, and clonogenicity in EAC cells. These changes were associated with concomitant increases in ATP, ROS, and lactate production indicative of increased mitochondrial respiration as well as glycolytic activity. DCA and CSC exposure significantly decreased expression of uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein implicated in regulation of the proton gradient. Knockdown of UCP2 in EACC phenocopied DCA and CSC exposure as evidenced by increased cell migration, invasion, and clonogenicity, whereas over-expression of UCP2 had an inverse effect. Furthermore, over-expression of UCP2 abrogated DCA and CSC-mediated increases in lactate and ATP production in EACC.DCA and CSC promote the aggressive phenotype of EACC with concomitant metabolic changes occurring via downregulation of UCP2. These results indicate that UCP2 is integral to the aggressive phenotype of EACC. This mechanism suggests that targeting alterations in cellular energetics may be a novel strategy for EAC therapy.

Leishmania donovani inhibits inflammasome-dependent macrophage activation by exploiting the negative regulatory proteins A20 and UCP2.

In visceral leishmaniasis, we found that the antileishmanial drug Amp B produces a higher level of IL-1β over the infected control. Moreover, administering anti-IL-1β antibody to infected Amp B-treated mice showed significantly less parasite clearance. Investigation revealed that Leishmania inhibits stimuli-induced expression of a multiprotein signaling platform, NLRP3 inflammasome, which in turn inhibits caspase-1 activation mediated maturation of IL-1β from its pro form. Attenuation of NLRP3 and pro-IL-1β in infection was found to result from decreased NF-κB activity. Transfecting infected cells with constitutively active NF-κB plasmid increased NLRP3 and pro-IL-1β expression but did not increase mature IL-1β, suggesting that IL-1β maturation requires a second signal, which was found to be reactive oxygen species (ROS). Decreased NF-κB was attributed to increased expression of A20, a negative regulator of NF-κB signaling. Silencing A20 in infected cells restored NLRP3 and pro-IL-1β expression, but also increased matured IL-1β, implying an NF-κB-independent A20-modulated IL-1β maturation. Macrophage ROS is primarily regulated by mitochondrial uncoupling protein 2 (UCP2), and UCP2-silenced infected cells showed an increased IL-1β level. Short hairpin RNA-mediated knockdown of A20 and UCP2 in infected mice independently documented decreased liver and spleen parasite burden and increased IL-1β production. These results suggest that Leishmania exploits A20 and UCP2 to impair inflammasome activation for disease propagation.-Gupta, A. K., Ghosh, K., Palit, S., Barua, J., Das, P. K., Ukil, A. Leishmania donovani inhibits inflammasome-dependent macrophage activation by exploiting the negative regulatory proteins A20 and UCP2.

Reactive oxygen species derived from NADPH oxidase 1 and mitochondria mediate angiotensin II-induced smooth muscle cell senescence

Cellular senescence has emerged as an important player in both physiology and pathology. Excessive reactive oxygen species (ROS) is known to mediate cellular senescence. NADPH oxidases are major sources for ROS production in the vascular wall; the roles of different NADPH oxidase isoforms in cellular senescence remain unclear, however. We investigated the roles of two NADPH oxidase isoforms in mitochondrial dysfunction during angiotensin II (Ang II)-induced cellular senescence of human aortic vascular smooth muscle cells (VSMCs). Ang II (10−7M) stimulated ROS generation, exhibiting early increases between 30 and 60min and sustained increases between 24h and 72h, and induced VSMCs senescence after 48h or 72h treatment as assessed with senescence-associated β-galactosidase activity and the expression of two cell cycle inhibitors, p21 and p16. ROS scavengers and membrane-permeable catalase (catalase-PEG) reduced Ang II-stimulated cellular senescence. Furthermore, small interfering RNA (siRNA) of NADPH oxidase catalytic subunit Nox1, but not that of another isoform Nox4, inhibited Ang II-induced cellular senescence. Nox1 siRNA inhibited both early and sustained ROS increases induced by Ang II. In addition, a mitochondrial-specific antioxidant, mitoQ10, effectively inhibited Ang II-induced ROS increases and cellular senescence. Ang II decreased ATP synthesis and induced mitochondrial membrane depolarization, which were attenuated by pre-treating cells with Nox1 siRNA, mitoQ10 or catalase-PEG. The effect of Ang II on the mitochondrial regulator peroxisome-proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its downstream genes was examined. Ang II stimulated S570 phosphorylation of PGC-1α with concomitant decreases in catalase and uncoupling protein-2 (UCP-2) levels between 12h and 72h, which were inhibited by Nox1 siRNA. Knockdown of both catalase and UCP-2 mimicked Ang II-induced VSMC senescence. These results suggested that Ang II-stimulated Nox1 activation mediates mitochondrial dysfunction, probably by decreasing PGC-1α activity and increasing mitochondrial oxidative stress, and leads to cellular senescence of VSMCs.

MiR-133a Is Functionally Involved in Doxorubicin-Resistance in Breast Cancer Cells MCF-7 via Its Regulation of the Expression of Uncoupling Protein 2.

The development of novel targeted therapies holds promise for conquering chemotherapy resistance, which is one of the major hurdles in current breast cancer treatment. Previous studies indicate that mitochondria uncoupling protein 2 (UCP-2) is involved in the development of chemotherapy resistance in colon cancer and lung cancer cells. In the present study we found that lower level of miR133a is accompanied by increased expression of UCP-2 in Doxorubicin-resistant breast cancer cell cline MCF-7/Dox as compared with its parental cell line MCF-7. We postulated that miR133a might play a functional role in the development of Doxorubicin-resistant in breast cancer cells. In this study we showed that: 1) exogenous expression of miR133a in MCF-7/Dox cells can sensitize their reaction to the treatment of Doxorubicin, which is coincided with reduced expression of UCP-2; 2) knockdown of UCP-2 in MCF-7/Dox cells can also sensitize their reaction to the treatment of Doxorubicin; 3) intratumoral delivering of miR133a can restore Doxorubicin treatment response in Doxorubicin-resistant xenografts in vivo, which is concomitant with the decreased expression of UCP-2. These findings provided direct evidences that the miR133a/UCP-2 axis might play an essential role in the development of Doxorubicin-resistance in breast cancer cells, suggesting that the miR133a/UCP-2 signaling cohort could be served as a novel therapeutic target for the treatment of chemotherapy resistant in breast cancer.

Stress-Induced Glucocorticoid Release Upregulates Uncoupling Protein-2 Expression and Enhances Resistance to Endotoxin-Induced Lethality

Objective: Although psychological and/or physiological stress has been well documented to influence immune responses, the precise mechanism for immunomodulation remains to be elucidated. The present work describes the role of the hypothalamic-pituitary-adrenal (HPA) axis in the mechanism of stress-mediated enhanced-resistance to lethality after lipopolysaccharide (LPS) injection. Methods/Results: Preconditioning with restraint stress (RS) resulted in enhanced activation of the HPA axis in response to LPS injection and suppressed LPS-induced release of proinflammatory cytokines and nitric oxide metabolites. Melanocortin 2 receptor-deficient mice (MC2R^-/-) failed to increase plasma levels of glucocorticoids in response to LPS injection, and exhibited high sensitivity to LPS-induced lethality with enhanced release of proinflammatory cytokines as compared with MC2R^+/- mice. Real-time PCR analysis revealed that RS induced upregulation of uncoupling protein-2 (UCP2) in macrophages in the lung and the liver of MC2R^+/-, but not of MC2R^-/-, mice. In addition, RS increased UCP2-dependent uncoupling activity of isolated mitochondria from the liver of MC2R^+/-, but not of MC2R^-/-, mice. In vitro study revealed that corticosterone and dexamethasone directly increased UCP2 expression in mouse RAW 264.7 macrophages and suppressed the generation of LPS-induced mitochondrial reactive oxygen species (ROS) and TNF-α production. Knockdown of UCP2 by small interfering RNA blunted the dexamethasone action for suppressing LPS-induced mitochondrial ROS and TNF-α production. Conclusion: The present work suggests that RS enhances activation of the HPA axis to release glucocorticoids and upregulation of UCP2 in macrophages, thereby increasing the resistance to endotoxin-induced systemic inflammation and death.

2014 Young Investigator Award of the American Physiological Society Renal Section

2014 Young Investigator Award of the American Physiological Society Renal Section

A microRNA-30e/mitochondrial uncoupling protein 2 axis mediates TGF-β1-induced tubular epithelial cell extracellular matrix production and kidney fibrosis

Mitochondria dysfunction has been reported in various kidney diseases but how it leads to kidney fibrosis and how this is regulated is unknown. Here we found that mitochondrial uncoupling protein 2 (UCP2) was induced in kidney tubular epithelial cells after unilateral ureteral obstruction in mice and that mice with ablated UCP2 resisted obstruction-induced kidney fibrosis. We tested this association further in cultured NRK-52E cells and found that TGF-β1 remarkably induced UCP2 expression. Knockdown of UCP2 largely abolished the effect of TGF-β1, whereas overexpression of UCP2 promoted tubular cell phenotype changes. Analysis using a UCP2 mRNA-3′-untranslated region luciferase construct showed that UCP2 mRNA is a direct target of miR-30e. MiR-30e was downregulated in tubular cells from fibrotic kidneys and TGF-β1-treated NRK-52E cells. A miR-30e mimic significantly inhibited TGF-β1-induced tubular-cell epithelial–mesenchymal transition, whereas a miR-30e inhibitor imitated TGF-β1 effects. Finally, genipin, an aglycone UCP2 inhibitor, significantly ameliorated kidney fibrosis in mice. Thus, the miR-30e/UCP2 axis has an important role in mediating TGF-β1-induced epithelial–mesenchymal transition and kidney fibrosis. Targeting this pathway may shed new light for the future of fibrotic kidney disease therapy.

Glutathionylation of UCP2 sensitizes drug resistant leukemia cells to chemotherapeutics

Uncoupling protein-2 (UCP2) is used by cells to control reactive oxygen species (ROS) production by mitochondria. This ability depends on the glutathionylation state of UCP2. UCP2 is often overexpressed in drug resistant cancer cells and therein controls cell ROS levels and limits drug toxicity. With our recent observation that glutathionylation deactivates proton leak through UCP2, we decided to test if diamide, a glutathionylation catalyst, can sensitize drug resistant cells to chemotherapeutic agents. Using drug sensitive HL-60 cells and the drug resistant HL-60 subline, Mx2, we show that chemical induction of glutathionylation selectively deactivates proton leak through UCP2 in Mx2 cells. Chemical glutathionylation of UCP2 disables chemoresistance in the Mx2 cells. Exposure to 200μM diamide led to a significant increase in Mx2 cell death that was augmented when cells were exposed to either menadione or the anthracycline doxorubicin. Diamide also sensitized Mx2 cells to a number of other chemotherapeutics. Proton leak through UCP2 contributed significantly to the energetics of the Mx2 cells. Knockdown of UCP2 led to a significant decrease in both resting and state 4 (i.e., proton leak-dependent) respiration (~43% and 62%, respectively) in Mx2 cells. Similarly diamide inhibited proton leak-dependent respiration by ~64%. In contrast, diamide had very little effect on proton leak in HL-60 cells. Collectively, our observations indicate that manipulation of UCP2 glutathionylation status can serve as a therapeutic strategy for cancer treatment.