n Hsien Chu

Akt mediates 17β-estradiol and/or estrogen receptor-α inhibition of LPS-induced tumor necresis factor-α expression and myocardial cell apoptosis by suppressing the JNK1/2-NFκB pathway

Evidence shows that women have lower tumour necrosis factor‐α (TNF‐α) levels and lower incidences of heart dysfunction and sepsis‐related morbidity and mortality. To identify the cardioprotective effects and precise cellular/molecular mechanisms behind estrogen and estrogen receptors (ERs), we investigated the effects of 17β‐estradiol (E2) and estrogen receptor α (ERα) on LPS‐induced apoptosis by analyzing the activation of survival and death signalling pathways in doxycycline (Dox)‐inducible Tet‐On/ERα H9c2 myocardial cells and ERα‐transfected primary cardiomyocytes overexpressing ERα. We found that LPS challenge activated JNK1/2, and then induced IκB degradation, NFκB activation, TNF‐α up‐regulation and subsequent myocardial apoptotic responses. In addition, treatments involving E2, membrane‐impermeable BSA‐E2 and/or Dox, which induces ERα overexpression, significantly inhibited LPS‐induced apoptosis by suppressing LPS‐up‐regulated JNK1/2 activity, IκB degradation, NFκB activation and pro‐apoptotic proteins (e.g. TNF‐α, active caspases‐8, t‐Bid, Bax, released cytochrome c, active caspase‐9, active caspase‐3) in myocardial cells. However, the cardioprotective properties of E2, BSA‐E2 and ERα overexpression to inhibit LPS‐induced apoptosis and promote cell survival were attenuated by applying LY294002 (PI3K inhibitor) and PI3K siRNA. These findings suggest that E2, BSA‐E2 and ERα expression exert their cardioprotective effects by inhibiting JNK1/2‐mediated LPS‐induced TNF‐α expression and cardiomyocyte apoptosis through activation of Akt.

IGF-II/mannose-6-phosphate receptor signaling induced cell hypertrophy and atrial natriuretic peptide/BNP expression via Gαq interaction and protein kinase C-α/CaMKII activation in H9c2 cardiomyoblast cells

The role played by IGF-II in signal transduction through the IGF-II/mannose-6-phosphate receptor (IGF2R) in heart tissue has been poorly understood. In our previous studies, we detected an increased expression of IGF-II and IGF2R in cardiomyocytes that had undergone pathological hypertrophy. We hypothesized that after binding with IGF-II, IGF2R may trigger intracellular signaling cascades involved in the progression of pathologically cardiac hypertrophy. In this study, we used immunohistochemical analysis of the human cardiovascular tissue array to detect expression of IGF2R. In our study of H9c2 cardiomyoblast cell cultures, we used the rhodamine phalloidin staining to measure the cell hypertrophy and western blot to measure the expression of cardiac hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cells treated with IGF-II. We found that a significant association between IGF2R overexpression and myocardial infarction. The treatment of H9c2 cardiomyoblast cells with IGF-II not only induced cell hypertrophy but also increased the protein level of ANP and BNP. Using Leu27IGF-II, an analog of IGF-II which interacts selectively with the IGF2R, to specifically activate IGF2R signaling cascades, we found that binding of Leu27IGF-II to IGF2R led to an increase in the phosphorylation of protein Kinase C (PKC)-alpha and calcium/calmodulin-dependent protein kinase II (CaMKII) in a Galphaq-dependent manner. By the inhibition of PKC-alpha/CaMKII activity, we found that IGF-II and Leu27IGF-II-induced cell hypertrophy and upregulation of ANP and BNP were significantly suppressed. Taken together, this study provides a new insight into the effects of the IGF2R and its downstream signaling in cardiac hypertrophy. The suppression of IGF2R signaling pathways may be a good strategy to prevent the progression of pathological hypertrophy.

Activation of Insulin-Like Growth Factor II Receptor Induces Mitochondrial-Dependent Apoptosis through Gαq and Downstream Calcineurin Signaling in Myocardial Cells

In previous studies, we have found that IGF-II and IGF-II receptor (IGF-IIR) dose dependently correlated with the progression of pathological hypertrophy after complete abdominal aorta ligation, which may play a critical role in angiotensin II-induced cardiomyocyte apoptosis. However, the detail mechanisms of IGF-IIR in the regulation of cell apoptosis in response to IGF-II remain unclear. By using IGF-IR short hairpin RNA to inhibit IGF-IR expression and using Leu27 IGF-II analog to activate specifically the IGF-IIR, we investigated the role of IGF-II/IGF-IIR activation and its downstream signaling. Our results revealed that IGF-II synergistically increased the cell apoptosis induced by suppressing of IGF-IR in neonatal rat ventricular myocytes. After binding of Leu27IGF-II, IGF-IIR became associated with alpha-q polypeptide, acted like a protein-coupled receptor to activate calcineurin, led to the translocation of Bad into mitochondria and release of cytochrome c into cytoplasm, and contributed to mitochondrial-dependent apoptosis in neonatal rat ventricular myocytes. Furthermore, inhibition of IGF-IIR, alpha-q polypeptide, or calcineurin by RNA interference could block the Leu27IGF-II-induced cell apoptosis. Together, this study provides a new insight into the effects of the IGF-IIR and its downstream signaling in myocardial apoptosis. Suppression of IGF-IIR signaling pathways may be a good strategy for both the protection against myocardial cell apoptosis and the prevention of heart failure progression.

A Placebo-Controlled Trial of Dextromethorphan as an Adjunct in Opioid-Dependent Patients Undergoing Methadone Maintenance Treatment

Background: Low-dose dextromethorphan (DM) might have anti-inflammatory and neurotrophic effects mechanistically remote from an NMDA receptor. In a randomized, double-blind, controlled 12 week study, we investigated whether add-on dextromethorphan reduced cytokine levels and benefitted opioid-dependent patients undergoing methadone maintenance therapy (MMT). Methods: Patients were randomly assigned to a group: DM60 (60mg/day dextromethorphan; n = 65), DM120 (120mg/day dextromethorphan; n = 65), or placebo (n = 66). Primary outcomes were the methadone dose required, plasma morphine level, and retention in treatment. Plasma tumor necrosis factor (TNF)-α, C-reactive protein, interleukin (IL)-6, IL-8, transforming growth factor–β1, and brain-derived neurotrophic factor (BDNF) levels were examined during weeks 0, 1, 4, 8, and 12. Multiple linear regressions with generalized estimating equation methods were used to examine the therapeutic effect. Results: After 12 weeks, the DM60 group had significantly longer treatment retention and lower plasma morphine levels than did the placebo group. Plasma TNF-α was significantly decreased in the DM60 group compared to the placebo group. However, changes in plasma cytokine levels, BDNF levels, and the methadone dose required in the three groups were not significantly different. Conclusions: We provide evidence—decreased concomitant heroin use—of low-dose add-on DM’s efficacy for treating opioid-dependent patients undergoing MMT.

Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91phox subunit of NADPH oxidase

BackgroundBoth (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner. Multiple lines of evidence have documented a pivotal role of overactivated NADPH oxidase (NOX2) in inflammation-mediated neurodegeneration. We hypothesized that NOX2 might be a novel action site of naloxone to mediate its anti-inflammatory actions.MethodsInhibition of NOX-2-derived superoxide by (-) and (+)-naloxone was measured in lipopolysaccharide (LPS)-treated midbrain neuron-glia cultures and phorbol myristate acetate (PMA)-stimulated neutrophil membranes by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt (WST-1) or ferricytochrome c. Further, various ligand (3H-naloxone) binding assays were performed in wild type and gp91phox-/- neutrophils and transfected COS-7 and HEK293 cells. The translocation of cytosolic subunit p47phox to plasma membrane was assessed by western blot.ResultsBoth (-) and (+)-naloxone equally inhibited LPS- and PMA-induced superoxide production with an IC50 of 1.96 and 2.52 μM, respectively. Competitive binding of 3H-naloxone with cold (-) and (+)-naloxone in microglia showed equal potency with an IC50 of 2.73 and 1.57 μM, respectively. 3H-Naloxone binding was elevated in COS-7 and HEK293 cells transfected with gp91phox; in contrast, reduced 3H-naloxone binding was found in neutrophils deficient in gp91phoxor in the presence of a NOX2 inhibitor. The specificity and an increase in binding capacity of 3H-naloxone were further demonstrated by 1) an immunoprecipitation study using gp91phoxantibody, and 2) activation of NOX2 by PMA. Finally, western blot studies showed that naloxone suppressed translocation of the cytosolic subunit p47phoxto the membrane, leading to NOX2 inactivation.ConclusionsStrong evidence is provided indicating that NOX2 is a non-opioid novel binding site for naloxone, which is critical in mediating its inhibitory effect on microglia overactivation and superoxide production.

α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation

Significance α-Synuclein (α-syn) aggregates released from neurons activate microglia, leading to chronic neuroinflammation that causes damage to neurons in brains with synucleinopathies, such as Parkinson’s disease (PD). However, little is known about the mechanism by which α-syn affects microglial activity, especially motility, and why microglia migrate toward the injured neurons and preferentially accumulate along with α-syn aggregates in the affected areas, e.g., in the substantia nigra of PD brains. Here we show that neuron-derived α-syn aggregates are chemoattractants that direct microglial migration by acting on NADPH oxidase and several specific downstream proteins. Blocking the targets involved in α-syn–mediated microglial directional migration may represent a therapeutic strategy to protect against progressive neuronal loss in PD and related synucleinopathies. Malformed α-Synuclein (α-syn) aggregates in neurons are released into the extracellular space, activating microglia to induce chronic neuroinflammation that further enhances neuronal damage in α-synucleinopathies, such as Parkinson’s disease. The mechanisms by which α-syn aggregates activate and recruit microglia remain unclear, however. Here we show that α-syn aggregates act as chemoattractants to direct microglia toward damaged neurons. In addition, we describe a mechanism underlying this directional migration of microglia. Specifically, chemotaxis occurs when α-syn binds to integrin CD11b, leading to H2O2 production by NADPH oxidase. H2O2 directly attracts microglia via a process in which extracellularly generated H2O2 diffuses into the cytoplasm and tyrosine protein kinase Lyn, phosphorylates the F-actin–associated protein cortactin after sensing changes in the microglial intracellular concentration of H2O2. Finally, phosphorylated cortactin mediates actin cytoskeleton rearrangement and facilitates directional cell migration. These findings have significant implications, given that α-syn–mediated microglial migration reaches beyond Parkinson’s disease.

ANG II promotes IGF-IIR expression and cardiomyocyte apoptosis by inhibiting HSF1 via JNK activation and SIRT1 degradation.

Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure. Our previous studies found that the activation of insulin-like growth factor receptor II (IGF-IIR) signaling was critical for hypertensive angiotensin II (ANG II)-induced cardiomyocyte apoptosis. However, the detailed mechanism by which ANG II regulates IGF-IIR in heart cells remains elusive. In this study, we found that ANG II activated its downstream kinase JNK to increase IGF-IIR expression through the ANG II receptor angiotensin type 1 receptor. JNK activation subsequently led to sirtuin 1 (SIRT1) degradation via the proteasome, thus preventing SIRT1 from deacetylating heat-shock transcription factor 1 (HSF1). The resulting increase in the acetylation of HSF1 impaired its ability to bind to the IGF-IIR promoter region (nt −748 to −585). HSF1 protected cardiomyocytes by acting as a repressor of IGF-IIR gene expression, and ANG II diminished this HSF1-mediated repression through enhanced acetylation, thus activating the IGF-IIR apoptosis pathway. Taken together, these results suggest that HSF1 represses IGF-IIR gene expression to protect cardiomyocytes. ANG II activates JNK to degrade SIRT1, resulting in HSF1 acetylation, which induces IGF-IIR expression and eventually results in cardiac hypertrophy and apoptosis. HSF1 could be a valuable target for developing treatments for cardiac diseases in hypertensive patients.

Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, protects dopaminergic neurons from neurotoxin-induced damage.

BACKGROUND AND PURPOSE Prevention or disease‐modifying therapies are critical for the treatment of neurodegenerative disorders such as Alzheimers disease, Parkinsons disease and Huntingtons disease. However, no such intervention is currently available. Growing evidence has demonstrated that administration of histone deacetylase (HDAC) inhibitors ameliorates a wide range of neurologic and psychiatric disorders in experimental models. Suberoylanilide hydroxamic acid (SAHA) was the first HDAC inhibitor approved by the Food and Drug Administration for the sole use of cancer therapy. The purpose of this study was to explore the potential new indications of SAHA for therapy of neurodegenerative diseases in in vitro Parkinsons disease models.

Histone Acetylation Is Essential for ANG-II-Induced IGF-IIR Gene Expression in H9c2 Cardiomyoblast Cells and Pathologically Hypertensive Rat Heart

The IGF‐II/mannose 6‐phosphate receptor (IGF‐IIR/Man‐6‐P) up‐regulation correlates with heart disease progression and its signaling cascades directly trigger pathological cardiac hypertrophy, fibrosis, and cardiomyocytes apoptosis. IGF‐IIR gene expression/ suppression is able to prevent myocardial remodeling. However, the regulating mechanisms for the IGF‐IIR gene remain unclear. This study performed reverse transcriptase PCR (RT‐PCR) and methylation‐specific PCR (MS‐PCR) to detect expression and DNA methylation of CpG islands within the IGF‐IIR genomic DNA region. Our finding revealed that the IGF‐IIR gene was up‐regulated both in H9c2 cells treated with tumor necrosis factor‐alpha (TNF‐α), lipopolysaccharide (LPS), angiotensin II (ANGII) and inomycin, and age‐dependently in spontaneously hypertensive rat (SHR) heart. For the DNA methylation study, although there were four CpG islands within IGF‐IIR genomic regions, the DNA methylation distribution showed no change either in cells treated with ANGII or in the SHR heart. Using chemical inhibitors to individually block histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity, we found that histone acetylation was essential for ANGII‐induced IGF‐IIR gene expression using RT‐PCR and luciferase assay. The Chromatin immuno‐precipitation assay indicated that acetyl‐Histone H3 and acetyl‐Histone H4 associated with the IGF‐IIR promoter increased in the presence of ANGII, otherwise methyl‐CpG binding domain protein 2 (MeCP2) is disassociated with this. Taken together, this study demonstrates that histone acetylation plays a critical role in IGF‐IIR up‐regulation during pathological cardiac diseases and might provide a targeting gene in transcriptional therapies for the failing heart. J. Cell. Physiol. 227: 259–268, 2012.

Lipopolysaccharide induces cellular hypertrophy through calcineurin/NFAT-3 signaling pathway in H9c2 myocardiac cells

Evidences suggest that lipopolysaccharide (LPS) participates in the inflammatory response in the cardiovascular system; however, it is unknown if LPS is sufficient to cause the cardiac hypertrophy. In the present study, we treated H9c2 myocardiac cells with LPS to explore whether LPS causes cardiac hypertrophy, and to identify the precise molecular and cellular mechanisms behind hypertrophic responses. Here we show that LPS challenge induces pathological hypertrophic responses such as the increase in cell size, the reorganization of actin filaments, and the upregulation of hypertrophy markers including atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in H9c2 cells. LPS treatment significantly promotes the activation of GATA-4 and the nuclear translocation of NFAT-3, which act as transcription factors mediating the development of cardiac hypertrophy. After administration of inhibitors including U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), CsA (calcineurin inhibitor), FK506 (calcineurin inhibitor), and QNZ (NFκB inhibitor), LPS-induced hypertrophic characteristic features, such as increases in cell size, actin fibers, and levels of ANP and BNP, and the nuclear localization of NFAT-3 are markedly inhibited only by calcineurin inhibitors, CsA and FK506. Collectively, these results suggest that LPS leads to myocardiac hypertrophy through calcineurin/NFAT-3 signaling pathway in H9c2 cells. Our findings further provide a link between the LPS-induced inflammatory response and the calcineurin/NFAT-3 signaling pathway that mediates the development of cardiac hypertrophy.