Youn Wook Chung

Selenoprotein W is a glutathione‐dependent antioxidant in vivo

The function of selenoprotein W (Se‐W) was investigated by cloning the corresponding cDNA from mouse brain and expressing it in CHO cells and H1299 human lung cancer cells. Overexpression of Se‐W markedly reduced the sensitivity of both cell lines to H2O2 cytotoxicity. The intracellular peroxide concentration of the transfected cells was lower than that of the parental cells in the absence or presence of extracellular H2O2. The resistance to oxidative stress conferred by Se‐W was dependent on glutathione. Expression of Se‐W mutants in which selenocysteine‐13 or cysteine‐37 was replaced by serine did not confer resistance to H2O2, implicating these residues in the antioxidant activity of Se‐W in vivo.

From PDE3B to the regulation of energy homeostasis

The incidence of obesity in the developed world is increasing at an alarming rate. Concurrent with the increase in the incidence of obesity is an increase in the incidence of type 2 diabetes. Cyclic AMP (cAMP) and cGMP are key second messengers in all cells; for example, when it comes to processes of relevance for the regulation of energy metabolism, cAMP is a key mediator in the regulation of lipolysis, glycogenolysis, gluconeogenesis and pancreatic β cell insulin secretion. PDE3B, one of several enzymes which hydrolyze cAMP and cGMP, is expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, hypothalamic cells and β cells. It has been shown, using PDE3 inhibitors and gene targeting approaches in cells and animals, that altered levels of PDE3B result in a number of changes in the regulation of glucose and lipid metabolism and in overall energy homeostasis. This article highlights the complexity involved in the regulation of PDE3B by hormones, and in the regulation of downstream metabolic effects by PDE3B in several interacting tissues.

Effect of progerin on the accumulation of oxidized proteins in fibroblasts from Hutchinson Gilford progeria patients.

The mutation responsible for Hutchinson Gilford Progeria Syndrome (HGPS) causes abnormal nuclear morphology. Previous studies show that free radicals and reactive oxygen species play major roles in the etiology and/or progression of neurodegenerative diseases and aging. This study compares oxidative stress responses between progeric and normal fibroblasts. Our data revealed higher ROS levels in HGPS cells compared to age-matched controls. In response to oxidative challenge, progeric cells showed increased mRNA levels for mitochondrial superoxide dismutase (SOD) and SOD protein content. However, this did not prevent a drop in the ATP content of progeria fibroblasts. Previous studies have shown that declines in human fibroblast ATP levels interfere with programmed cell death and promote necrotic inflammation. Notably, in our investigations the ATP content of progeria fibroblasts was only approximately 50% of that found in healthy controls. Furthermore, HGPS fibroblast analysis revealed a decrease in total caspase-like proteasome activity and in the levels of two active proteolytic complex subunits (beta(5) and beta(7)). A number of studies indicate that the molecular mechanisms causing accelerated aging in progeric patients also occur in healthy cells of older individuals. Thus, the results of this study may also help explain some of the cellular changes that accompany normal aging.

H2O2-induced AP-1 activation and its effect on p21waf1/cip1-mediated G2/M arrest in a p53-deficient human lung cancer cell

Cellular response to oxidative stress is a complex process that is often connected to cell cycle regulation. The present study examines the effect of H(2)O(2) on cell cycle regulation and involvement of reactive oxygen species (ROS) in these H(2)O(2)-induced responses in a p53-deficient human lung carcinoma cell line, H1299. Treatment of the cells with H(2)O(2) caused a G2/M phase arrest. Among the redox-sensitive transcription factors, NF-kappaB and AP-1, we found that only AP-1 was activated by 200 microM H(2)O(2) in human lung cells. Furthermore, electrophoretic mobility shift assays revealed that H(2)O(2) enhanced the DNA binding of AP-1 to a putative AP-1 binding element (TGAGGAA) in the p21(WAF1/CIP1) promoter region (between -2203 and -2197 nucleotides upstream of the transcription initiation site). An increase in c-Jun phosphorylation by ERK was also found to accompany the increased AP-1 activity as detected by Western blot. PD98059, a specific inhibitor of MEK, diminished H(2)O(2)-induced phosphorylation of c-Jun and DNA binding activity of AP-1, decreased expression of p21(WAF1/CIP1), and released the cells from G2/M arrest. Taken together, these results revealed a novel AP-1 binding site in the promoter region of p21(WAF1/CIP1) and a possible cell cycle regulation mechanism mediated by activation of a redox-dependent ERK signaling pathway.

Phosphodiesterase Type 3A Regulates Basal Myocardial Contractility Through Interacting With Sarcoplasmic Reticulum Calcium ATPase Type 2a Signaling Complexes in Mouse Heart

Rationale: cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A vs PDE3B, in the regulation of heart function remains unclear. Objective: To determine the relative contribution of PDE3A vs PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. Methods and Results: Compared with wild-type littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A−/−, but not PDE3B−/−, mice. Furthermore, PDE3 inhibition had no effect on PDE3A−/− hearts but increased contractility in wild-type (as expected) and PDE3B−/− hearts to levels indistinguishable from PDE3A−/−. The enhanced contractility in PDE3A−/− hearts was associated with cAMP-dependent elevations in Ca2+ transient amplitudes and increased sarcoplasmic reticulum (SR) Ca2+ content, without changes in L-type Ca2+ currents of cardiomyocytes, as well as with increased SR Ca2+-ATPase type 2a activity, SR Ca2+ uptake rates, and phospholamban phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ≈8-fold in SR fractions from PDE3A−/− hearts. Coimmunoprecipitation experiments further revealed that PDE3A associates with both SR calcium ATPase type 2a and phospholamban in a complex that also contains A-kinase anchoring protein-18, protein kinase type A-RII, and protein phosphatase type 2A. Conclusions: Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca2+ content by regulating cAMP in microdomains containing macromolecular complexes of SR calcium ATPase type 2a–phospholamban–PDE3A.Rationale: cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A versus PDE3B, in the regulation of heart function remains unclear. Objective: To determine the relative contribution of PDE3A versus PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. Methods and Results: Compared to wild-type (WT) littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A -/- , but not PDE3B -/- , mice. Furthermore, PDE3 inhibition had no effect on PDE3A -/- hearts but increased contractility in WT (as expected) and PDE3B -/- hearts to levels indistinguishable from PDE3A -/- . The enhanced contractility in PDE3A -/- hearts was associated with cAMP-dependent elevations in Ca 2+ transient amplitudes and increased SR Ca 2+ content, without changes in L-type Ca 2+ currents (I CaL ) of cardiomyocytes, as well as with increased SR Ca 2+ -ATPase (SERCA2a) activity, SR Ca 2+ uptake rates, and phospholamban (PLN) phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ~8-fold in SR fractions from PDE3A -/- hearts. Co-immunoprecipitation experiments further revealed that PDE3A associates with both SERCA2a and PLN in a complex which also contains AKAP-18, PKA-RII and PP2A. Conclusions: Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca 2+ content by regulating cAMP in microdomains containing macromolecular complexes of SERCA2a-PLN-PDE3A.

PDE3A Regulates Basal Myocardial Contractility Through Interacting with SERCA2a-Signaling Complexes in Mouse Heart

Rationale: cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A vs PDE3B, in the regulation of heart function remains unclear. Objective: To determine the relative contribution of PDE3A vs PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. Methods and Results: Compared with wild-type littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A−/−, but not PDE3B−/−, mice. Furthermore, PDE3 inhibition had no effect on PDE3A−/− hearts but increased contractility in wild-type (as expected) and PDE3B−/− hearts to levels indistinguishable from PDE3A−/−. The enhanced contractility in PDE3A−/− hearts was associated with cAMP-dependent elevations in Ca2+ transient amplitudes and increased sarcoplasmic reticulum (SR) Ca2+ content, without changes in L-type Ca2+ currents of cardiomyocytes, as well as with increased SR Ca2+-ATPase type 2a activity, SR Ca2+ uptake rates, and phospholamban phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ≈8-fold in SR fractions from PDE3A−/− hearts. Coimmunoprecipitation experiments further revealed that PDE3A associates with both SR calcium ATPase type 2a and phospholamban in a complex that also contains A-kinase anchoring protein-18, protein kinase type A-RII, and protein phosphatase type 2A. Conclusions: Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca2+ content by regulating cAMP in microdomains containing macromolecular complexes of SR calcium ATPase type 2a–phospholamban–PDE3A.Rationale: cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A versus PDE3B, in the regulation of heart function remains unclear. Objective: To determine the relative contribution of PDE3A versus PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. Methods and Results: Compared to wild-type (WT) littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A -/- , but not PDE3B -/- , mice. Furthermore, PDE3 inhibition had no effect on PDE3A -/- hearts but increased contractility in WT (as expected) and PDE3B -/- hearts to levels indistinguishable from PDE3A -/- . The enhanced contractility in PDE3A -/- hearts was associated with cAMP-dependent elevations in Ca 2+ transient amplitudes and increased SR Ca 2+ content, without changes in L-type Ca 2+ currents (I CaL ) of cardiomyocytes, as well as with increased SR Ca 2+ -ATPase (SERCA2a) activity, SR Ca 2+ uptake rates, and phospholamban (PLN) phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ~8-fold in SR fractions from PDE3A -/- hearts. Co-immunoprecipitation experiments further revealed that PDE3A associates with both SERCA2a and PLN in a complex which also contains AKAP-18, PKA-RII and PP2A. Conclusions: Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca 2+ content by regulating cAMP in microdomains containing macromolecular complexes of SERCA2a-PLN-PDE3A.

Dual Function of Protein Kinase C (PKC) in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced Manganese Superoxide Dismutase (MnSOD) Expression ACTIVATION OF CREB AND FOXO3a BY PKC-α PHOSPHORYLATION AND BY PKC-MEDIATED INACTIVATION OF Akt, RESPECTIVELY

12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA in human lung carcinoma cells, A549, mediated by a protein kinase C (PKC)-dependent activation of cAMP-responsive element-binding protein (CREB)-1/ATF-1-like factors. In this study, we showed that MnSOD protein expression was elevated in response to TPA or TNF-α, but not to hydrogen peroxide treatment. TPA-induced generation of reactive oxygen species (ROS) was blocked by pretreatment of the PKC inhibitor BIM and NADPH oxidase inhibitor DPI. Small interfering RNA (siRNA) experiments indicated that knocking down the NADPH oxidase components e.g. Rac1, p22phox, p67phox, and NOXO1 in A549 cells impaired TPA-induced MnSOD expression. To identify the PKC isozyme involved, we used a sod2 gene response reporter plasmid, pSODLUC-3340-I2E-C, capable of sensing the effect of TNF-α and TPA, to monitor the effects of PKC isozyme-specific inhibitors and siRNA-induced knockdown of specific PKC isozyme. Our data indicate that TPA-induced MnSOD expression was independent of p53 and most likely mediated by PKC-α-, and -ϵ-dependent signaling pathways. Furthermore, siRNA-induced knock-down of CREB and Forkhead box class O (FOXO) 3a led to a reduction in TPA-induced MnSOD gene expression. Together, our results revealed that TPA up-regulates, in part, two PKC-dependent transcriptional pathways to induce MnSOD expression. One pathway involves PKC-α catalyzed phosphorylation of CREB and the other involves a PKC-mediated the PP2A catalyzed dephosphorylation of Akt at Ser473 which in turn leads to FOXO3a Ser253 dephosphorylation and its activation.

Antioxidative role of selenoprotein W in oxidant-induced mouse embryonic neuronal cell death

It has been reported that selenoprotein W (SelW) mRNA is highly expressed in the developing central nerve system of rats, and its expression is maintained until the early postnatal stage. We here found that SelW protein significantly increased in mouse brains of postnatal day 8 and 20 relative to embryonic day 15. This was accompanied by increased expression of SOD1 and SOD2. When the expression of SelW in primary cultured cells derived from embryonic cerebral cortex was knocked down with small interfering RNAs (siRNAs), SelW siRNA-transfected neuronal cells were more sensitive to the oxidative stress induced by treatment of H2O2 than control cells. TUNEL assays revealed that H2O2-induced apoptotic cell death occurred at a higher frequency in the siRNA-transfected cells than in the control cells. Taken together, our findings suggest that SelW plays an important role in protection of neurons from oxidative stress during neuronal development.

Targeted disruption of PDE3B, but not PDE3A, protects murine heart from ischemia/reperfusion injury

Significance By catalyzing the destruction of cAMP and cGMP, cyclic nucleotide phosphodiesterases (PDEs) regulate their intracellular concentrations and biological actions. Eleven distinct gene families (PDE1–PDE11) define the PDE superfamily. Most families contain several PDE genes. Two separate but related genes generate PDE3 subfamilies PDE3A and PDE3B. Although inhibition of PDE3 protects rodent heart against ischemia/reperfusion (I/R) injury, the specific PDE3 isoform involved is undetermined. Using PDE3A- and PDE3B-KO mice, we report that deletion of PDE3B, but not PDE3A, protected mouse heart from I/R injury in vivo and in vitro, via cAMP-induced preconditioning. To our knowledge, our study is the first to define a role for PDE3B in cardioprotection against I/R injury and suggests PDE3B as a target for cardiovascular therapies. Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B−/− heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B−/− mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse tubules where PDE3B was localized with caveolin-3. Moreover, a PDE3B−/− mitochondrial fraction containing connexin-43 and caveolin-3 was more resistant to Ca2+-induced opening of the mitochondrial permeability transition pore. Proteomics analyses indicated that PDE3B−/− heart mitochondria fractions were enriched in buoyant ischemia-induced caveolin-3–enriched fractions (ICEFs) containing cardioprotective proteins. Accumulation of proteins into ICEFs was PKA dependent and was achieved by ischemic preconditioning or treatment of WT heart with the PDE3 inhibitor cilostamide. Taken together, these findings indicate that PDE3B deletion confers cardioprotective effects because of cAMP/PKA-induced preconditioning, which is associated with the accumulation of proteins with cardioprotective function in ICEFs. To our knowledge, our study is the first to define a role for PDE3B in cardioprotection against I/R injury and suggests PDE3B as a target for cardiovascular therapies.

Dual function of protein kinase C in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MnSOD expression: Activation of CREB and FOXO3a by PKC-α phosphorylation and by PKC-mediated Inactivation of Akt, respectively

12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA in human lung carcinoma cells, A549, mediated by a protein kinase C (PKC)-dependent activation of cAMP-responsive element-binding protein (CREB)-1/ATF-1-like factors. In this study, we showed that MnSOD protein expression was elevated in response to TPA or TNF-α, but not to hydrogen peroxide treatment. TPA-induced generation of reactive oxygen species (ROS) was blocked by pretreatment of the PKC inhibitor BIM and NADPH oxidase inhibitor DPI. Small interfering RNA (siRNA) experiments indicated that knocking down the NADPH oxidase components e.g. Rac1, p22phox, p67phox, and NOXO1 in A549 cells impaired TPA-induced MnSOD expression. To identify the PKC isozyme involved, we used a sod2 gene response reporter plasmid, pSODLUC-3340-I2E-C, capable of sensing the effect of TNF-α and TPA, to monitor the effects of PKC isozyme-specific inhibitors and siRNA-induced knockdown of specific PKC isozyme. Our data indicate that TPA-induced MnSOD expression was independent of p53 and most likely mediated by PKC-α-, and -ϵ-dependent signaling pathways. Furthermore, siRNA-induced knock-down of CREB and Forkhead box class O (FOXO) 3a led to a reduction in TPA-induced MnSOD gene expression. Together, our results revealed that TPA up-regulates, in part, two PKC-dependent transcriptional pathways to induce MnSOD expression. One pathway involves PKC-α catalyzed phosphorylation of CREB and the other involves a PKC-mediated the PP2A catalyzed dephosphorylation of Akt at Ser473 which in turn leads to FOXO3a Ser253 dephosphorylation and its activation.