Insug Kang

AMP-activated Protein Kinase Activity Is Critical for Hypoxia-inducible Factor-1 Transcriptional Activity and Its Target Gene Expression under Hypoxic Conditions in DU145 Cells

AMP-activated protein kinase (AMPK) functions as an energy sensor to provide metabolic adaptations under the ATP-deprived conditions such as hypoxia. In the present study, we considered a role of AMPK in the adaptive response to hypoxia by examining whether AMPK is involved in the regulation of hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor that is critical for hypoxic induction of physiologically important genes. We demonstrate that hypoxia or CoCl2 rapidly activated AMPK in DU145 human prostate cancer cells, and its activation preceded the induction of HIF-1α expression. Under these conditions, blockade of AMPK activity by a pharmacological or molecular approach significantly attenuated hypoxia-induced responses such as HIF-1 target gene expression, secretion of vascular endothelial growth factor, glucose uptake, and HIF-1-dependent reporter gene expression, indicating that AMPK is critical for the HIF-1 transcriptional activity and its target gene expression. Its functional requirement for HIF-1 activity was also demonstrated in several different cancer cell lines, but AMPK activation alone was not sufficient to stimulate the HIF-1 transcriptional activity. We further present data showing that AMPK transmits a positive signal for HIF-1 activity via a signaling pathway that is independent of phosphatidylinositol 3-kinase/AKT and several mitogen-activated protein kinases. Taken together, our results suggest that AMPK is a novel and critical component of HIF-1 regulation, implying its new roles in oxygen-regulated cellular phenomena.

ANTI-INFLAMMATORY EFFECTS OF CROCIN AND CROCETIN IN RAT BRAIN MICROGLIAL CELLS

Microglial cells play critical roles in the immune and inflammatory responses of the central nervous system (CNS). Under pathological conditions, the activation of microglia helps in restoring CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that observed in Alzheimers and Parkinsons diseases. Crocin and crocetin, found in the fruits of gardenia and in the stigmas of saffron, have been considered for the treatment of various disorders in traditional oriental medicine. Crocin and crocetin have been reported to have diverse pharmacological functions, such as anti-hyperlipidemic, anti-atherosclerotic, and anti-cancer effects. Specifically, the neuroprotective potential of crocetin derivatives has previously been demonstrated. The specific aim of this study was to examine whether crocin or crocetin represses microglial activation. Crocin and crocetin were shown to be effective in the inhibition of LPS-induced nitric oxide (NO) release from cultured rat brain microglial cells. These compounds reduced the LPS-stimulated productions of tumor necrosis factor-α, interleukin-1β, and intracellular reactive oxygen species. The compounds also effectively reduced LPS-elicited NF-κB activation. In addition, crocin reduced NO release from microglia stimulated with interferon-γ and amyloid-β. In organotypic hippocampal slice cultures, both crocin and crocetin blocked the effect of LPS on hippocampal cell death. These results suggest that crocin and crocetin provide neuroprotection by reducing the production of various neurotoxic molecules from activated microglia.

Reactive oxygen species stabilize hypoxia-inducible factor-1 alpha protein and stimulate transcriptional activity via AMP-activated protein kinase in DU145 human prostate cancer cells

Hypoxia-inducible factor (HIF-1) plays a central role in the cellular adaptive response to hypoxic conditions, which are closely related to pathophysiological conditions, such as cancer. Although reactive oxygen species (ROS) have been implicated in the regulation of hypoxic and non-hypoxic induction of HIF-1 under various conditions, the role of ROS is quite controversial, and the mechanism underlying the HIF-1 regulation by ROS is not completely understood yet. Here, we investigated the biochemical mechanism for the ROS-induced HIF-1 by revealing a novel role of adenosine monophosphate-activated protein kinase (AMPK) and the upstream signal components. AMPK plays an essential role as energy-sensor under adenosine triphosphate-deprived conditions. Here we report that ROS induced by a direct application of H(2)O(2) and menadione to DU145 human prostate carcinoma resulted in accumulation of HIF-1alpha protein by attenuation of its degradation and activation of its transcriptional activity in an AMPK-dependent manner. By way of contrast, AMPK was required only for the transcriptional activity of HIF-1 under hypoxic condition, revealing a differential role of AMPK in these two stimuli. Furthermore, our data show that inhibition of AMPK enhances HIF-1alpha ubiquitination under ROS condition. Finally, we show that the regulation of HIF-1 by AMPK in response to ROS is under the control of c-Jun N-terminal kinase and Janus kinase 2 pathways. Collectively, our findings identify AMPK as a key determinant of HIF-1 functions in response to ROS and its possible role in the sophisticated HIF-1 regulatory mechanisms.

Activation of adenosine A3 receptor suppresses lipopolysaccharide-induced TNF-α production through inhibition of PI 3-kinase/Akt and NF-κB activation in murine BV2 microglial cells

Adenosine is an endogenous nucleoside that regulates many processes, including inflammatory responses, through activation of its receptors. Adenosine receptors have been reported to be expressed in microglia, which are major immune cells of brain, yet little is known about the role of adenosine receptors in microglial cytokine production. Thus, we investigated the effect of adenosine and adenosine A3 receptor ligands on LPS-induced tumor necrosis factor (TNF-alpha) production and its molecular mechanism in mouse BV2 microglial cells. Adenosine and Cl-IB-MECA, a specific adenosine A3 receptor agonist, suppressed LPS-induced TNF-alpha protein and mRNA levels. Moreover, MRS1523, a selective A3 receptor antagonist, blocked suppressive effects of both adenosine and Cl-IB-MECA on TNF-alpha. We further examined the effect of adenosine on signaling molecules, such as PI 3-kinase, Akt, p38, ERK1/2, and NF-kappaB, which are involved in the regulation of inflammatory responses. Adenosine inhibited LPS-induced phosphatidylinositol (PI) 3-kinase activation and Akt phosphorylation, whereas it had no effect on the phosphorylation of p38 and ERK1/2. We also found that adenosine as well as Cl-IB-MECA inhibited LPS-induced NF-kappaB DNA binding and luciferase reporter activity. Taken together, these results suggest that adenosine A3 receptor activation suppresses TNF-alpha production by inhibiting PI 3-kinase/Akt and NF-kappaB activation in LPS-treated BV2 microglial cells.

Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways.

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.

5-Aminoimidazole-4-carboxamide-ribonucleoside enhances oxidative stress-induced apoptosis through activation of nuclear factor-κB in mouse Neuro 2a neuroblastoma cells

AMP-activated protein kinase (AMPK) was recently suggested to have a pro-apoptotic effect although its primary function is believed to mediate cellular adaptation to metabolic stresses. Here, we investigated the effect of the AMPK activator 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) on oxidative stress-induced apoptosis using mouse Neuro 2a neuroblastoma cells. H2O2-induced apoptosis was increased by AMPK activation, either with AICAR pretreatment or with overexpression of active AMPK. AICAR also induced nuclear factor-kappaB (NF-kappaB) activation along with activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. Correlation between NF-kappaB activation and the AICAR-enhanced apoptotic cell death was observed. In addition, NF-kappaB inhibitor SN50 prevented the augmented cell death by AICAR. Thus, our data suggest that NF-kappaB mediates the pro-apoptotic effect of AICAR.

Overexpressed cyclophilin B suppresses apoptosis associated with ROS and Ca2+ homeostasis after ER stress.

Prolonged accumulation of misfolded proteins in the endoplasmic reticulum (ER) results in ER stress-mediated apoptosis. Cyclophilins are protein chaperones that accelerate the rate of protein folding through their peptidyl-prolyl cis-trans isomerase (PPIase) activity. In this study, we demonstrated that ER stress activates the expression of the ER-localized cyclophilin B (CypB) gene through a novel ER stress response element. Overexpression of wild-type CypB attenuated ER stress-induced cell death, whereas overexpression of an isomerase activity-defective mutant, CypB/R62A, not only increased Ca2+ leakage from the ER and ROS generation, but also decreased mitochondrial membrane potential, resulting in cell death following exposure to ER stress-inducing agents. siRNA-mediated inhibition of CypB expression rendered cells more vulnerable to ER stress. Finally, CypB interacted with the ER stress-related chaperones, Bip and Grp94. Taken together, we concluded that CypB performs a crucial function in protecting cells against ER stress via its PPIase activity.

Baicalein protects HT22 murine hippocampal neuronal cells against endoplasmic reticulum stress-induced apoptosis through inhibition of reactive oxygen species production and CHOP induction

Baicalein is one of the major flavonoids in Scutellaria baicalensis Georgi and possesses various effects, including cytoprotection and anti-inflammation. Because endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, such as Alzheimers disease, Parkinsons disease, and cerebral ischemia, we investigated the effects of baicalein on apoptotic death of HT22 mouse hippocampal neuronal cells induced by thapsigargin (TG) and brefeldin A (BFA), two representative ER stress inducers. Apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were measured by flow cytometry. Expression level and phosphorylation status of ER stress-associated proteins and activation and cleavage of apoptosis-associated proteins were analyzed by Western blot. Baicalein reduced TG- and BFA-induced apoptosis of HT22 cells and activation and cleavage of apoptosis-associated proteins, such as caspase-12 and -3 and poly(ADP-ribose) polymerase. Baicalein also reduced the TG- and BFA-induced expression of ER stress-associated proteins, including C/EBP homologous protein (CHOP) and glucose-regulated protein 78, the cleavage of X-box binding protein-1 and activating transcription factor 6α, and the phosphorylation of eukaryotic initiation factor-2α and mitogen-activated protein kinases, such as p38, JNK, and ERK. Knock-down of CHOP expression by siRNA transfection and specific inhibitors of p38 (SB203580), JNK (SP600125), and ERK (PD98059) as well as anti-oxidant (N-acetylcysteine) reduced TG- or BFA-induced cell death. Baicalein also reduced TG- and BFA-induced ROS accumulation and MMP reduction. Taken together, these results suggest that baicalein could protect HT22 neuronal cells against ER stress-induced apoptosis by reducing CHOP induction as well as ROS accumulation and mitochondrial damage.

Oleic acid reduces lipopolysaccharide-induced expression of iNOS and COX-2 in BV2 murine microglial cells: Possible involvement of reactive oxygen species, p38 MAPK, and IKK/NF-κB signaling pathways

Microglia are the major cells involved in neuroinflammation resulting in brain tissue damage during infection and neurodegenerative diseases. In this study, we examined the effects of the monounsaturated fatty acid oleic acid (OA) on LPS-induced proinflammatory mediators production and the mechanisms involved in BV2 microglia. OA inhibited LPS-induced expression of iNOS and COX-2 as well as production of NO and prostaglandin E2. We showed that OA blocked LPS-induced NF-kappaB activation and phosphorylation of inhibitor kappaB kinase (IKK). We also showed that OA inhibited LPS-induced phosphorylation of Akt and p38 MAPK, but not that of ERK. Finally, we showed that OA reduced reactive oxygen species (ROS) accumulation and an anti-oxidant N-acetylcysteine inhibited NF-kappaB transactivation and phosphorylation of IKK and Akt in LPS-stimulated BV2 cells. Taken together, our results suggest that OA shows an anti-inflammatory effect by inhibiting ROS, p38 MAPK, and Akt/IKK/NF-kappaB signaling pathways in LPS-stimulated BV2 microglia.

Melatonin stimulates glucose transport via insulin receptor substrate-1/phosphatidylinositol 3-kinase pathway in C2C12 murine skeletal muscle cells

Abstract:  The prevalence of diabetes has exponentially increased in recent decades due to environmental factors such as nocturnal lifestyle and aging, both of which influence the amount of melatonin produced in the pineal gland. The present study investigated the effect of melatonin on signaling pathways of glucose transport in C2C12 mouse skeletal muscle cells. Intriguingly, treatment of C2C12 cells with melatonin (1 nm) stimulated glucose uptake twofold increase. Melatonin‐stimulated glucose transport was inhibited with co‐treatment with the melatonin receptor antagonist luzindole. Furthermore, treatment of stably over‐expressed melatonin receptor type 2B containing C2C12 myotubes with melatonin amplified glucose transport c. 13‐fold. Melatonin also increased the phosphorylation level of insulin receptor substrate‐1 (IRS‐1) and the activity of phosphoinositide 3‐kinase (PI‐3‐kinase). However, 3′,5′‐cyclic adenosine monophosphate‐activated protein kinase (AMPK), another important glucose transport stimulatory mediator via an insulin‐independent pathway, was not influenced by melatonin treatment. Activity of p38 mitogen‐activated protein kinase (MAPK), a downstream mediator of AMPK, was also not changed by melatonin. In addition, melatonin increased the expression level of forkhead box A2, which was recently discovered to regulate fatty acid oxidation and to be inhibited by insulin. In summary, melatonin stimulates glucose transport to skeletal muscle cells via IRS‐1/PI‐3‐kinase pathway, which implies, at the molecular level, its role in glucose homeostasis and possibly in diabetes. Additionally, exposure to light at night and aging, both of which lower endogenous melatonin levels may contribute to the incidence and/or development of diabetes.