CD38/ADP-ribose/TRPM2-mediated nuclear Ca2+ signaling is essential for hepatic gluconeogenesis in fasting and diabetes.

Hepatic glucose production by glucagon is crucial for glucose homeostasis during fasting, yet the underlying mechanisms remain incompletely delineated. Although CD38 has been detected in the nucleus, its function in this compartment is unknown. Here, we demonstrate that nuclear CD38 (nCD38) controls glucagon-induced gluconeogenesis in primary hepatocytes and liver in a manner distinct from CD38 occurring in the cytoplasm and lysosomal compartments. We found that the localization of CD38 in the nucleus is required for glucose production by glucagon and that nCD38 activation requires NAD+ supplied by PKCδ-phosphorylated connexin 43. In fasting and diabetes, nCD38 promotes sustained Ca2+ signals via transient receptor potential melastatin 2 (TRPM2) activation by ADP-ribose, which enhances the transcription of glucose-6 phosphatase and phosphoenolpyruvate carboxykinase 1. These findings shed light on the role of nCD38 in glucagon-induced gluconeogenesis and provide insight into nuclear Ca2+ signals that mediate the transcription of key genes in gluconeogenesis under physiological conditions.

Activation of ROS-PERK-TFEB by filbertone ameliorates neurodegenerative diseases via enhancing the autophagy-lysosomal pathway.

The molecular mechanisms underlying the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease (PD), and Huntington's disease remain enigmatic, resulting in an unmet need for therapeutics development. Here, we suggest that filbertone, a key flavor compound found in the fruits of hazel trees of the genus Corylus, can ameliorate PD via lowering the abundance of aggregated α-synuclein. We previously reported that inhibition of hypothalamic inflammation by filbertone is mediated by suppression of nuclear factor kappa-B. Here, we report that filbertone activates PERK through mitochondrial reactive oxygen species production, resulting in the increased nuclear translocation of transcription factor-EB in SH-SY5Y human neuroblastoma cells. TFEB activation by filbertone promotes the autophagy-lysosomal pathway, which in turn alleviates the accumulation of α-synuclein. We also demonstrate that filbertone prevented the loss of dopaminergic neurons in the substantia nigra and striatum of mice on high-fat diet. Filbertone treatment also reduced high-fat diet-induced α-synuclein accumulation through upregulation of the autophagy-lysosomal pathway. In addition, filbertone improved behavioral abnormalities (i.e., latency time to fall and decrease of running distance) in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD murine model. In conclusion, filbertone may show promise as a potential therapeutic for neurodegenerative disease.

Phosphorylation of EIF2S1 (eukaryotic translation initiation factor 2 subunit alpha) is indispensable for nuclear translocation of TFEB and TFE3 during ER stress.

There are diverse links between macroautophagy/autophagy pathways and unfolded protein response (UPR) pathways under endoplasmic reticulum (ER) stress conditions to restore ER homeostasis. Phosphorylation of EIF2S1/eIF2α is an important mechanism that can regulate all three UPR pathways through transcriptional and translational reprogramming to maintain cellular homeostasis and overcome cellular stresses. In this study, to investigate the roles of EIF2S1 phosphorylation in regulation of autophagy during ER stress, we used EIF2S1 phosphorylation-deficient (A/A) cells in which residue 51 was mutated from serine to alanine. A/A cells exhibited defects in several steps of autophagic processes (such as autophagosome and autolysosome formation) that are regulated by the transcriptional activities of the autophagy master transcription factors TFEB and TFE3 under ER stress conditions. EIF2S1 phosphorylation was required for nuclear translocation of TFEB and TFE3 during ER stress. In addition, EIF2AK3/PERK, PPP3/calcineurin-mediated dephosphorylation of TFEB and TFE3, and YWHA/14-3-3 dissociation were required for their nuclear translocation, but were insufficient to induce their nuclear retention during ER stress. Overexpression of the activated ATF6/ATF6α form, XBP1s, and ATF4 differentially rescued defects of TFEB and TFE3 nuclear translocation in A/A cells during ER stress. Consequently, overexpression of the activated ATF6 or TFEB form more efficiently rescued autophagic defects, although XBP1s and ATF4 also displayed an ability to restore autophagy in A/A cells during ER stress. Our results suggest that EIF2S1 phosphorylation is important for autophagy and UPR pathways, to restore ER homeostasis and reveal how EIF2S1 phosphorylation connects UPR pathways to autophagy.Abbreviations: A/A: EIF2S1 phosphorylation-deficient; ACTB: actin beta; Ad-: adenovirus-; ATF6: activating transcription factor 6; ATZ: SERPINA1/α1-antitrypsin with an E342K (Z) mutation; Baf A1: bafilomycin A1; BSA: bovine serum albumin; CDK4: cyclin dependent kinase 4; CDK6: cyclin dependent kinase 6; CHX: cycloheximide; CLEAR: coordinated lysosomal expression and regulation; Co-IP: coimmunoprecipitation; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; EBSS: Earle's Balanced Salt Solution; EGFP: enhanced green fluorescent protein; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERAD: endoplasmic reticulum-associated degradation; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FBS: fetal bovine serum; gRNA: guide RNA; GSK3B/GSK3ß: glycogen synthase kinase 3 beta; HA: hemagglutinin; Hep: immortalized hepatocyte; IF: immunofluorescence; IRES: internal ribosome entry site; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LMB: leptomycin B; LPS: lipopolysaccharide; MAP1LC3A/B/LC3A/B: microtubule associated protein 1 light chain 3 alpha/beta; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; MFI: mean fluorescence intensity; MTORC1: mechanistic target of rapamycin kinase complex 1; NES: nuclear export signal; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; OE: overexpression; PBS: phosphate-buffered saline; PLA: proximity ligation assay; PPP3/calcineurin: protein phosphatase 3; PTM: post-translational modification; SDS: sodium dodecyl sulfate; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: standard error of the mean; TEM: transmission electron microscopy; TFE3: transcription factor E3; TFEB: transcription factor EB; TFs: transcription factors; Tg: thapsigargin; Tm: tunicamycin; UPR: unfolded protein response; WB: western blot; WT: wild-type; Xbp1s: spliced Xbp1; XPO1/CRM1: exportin 1.

PERK activation by SB202190 ameliorates amyloidogenesis via the TFEB-induced autophagy-lysosomal pathway.

The protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK), a key ER stress sensor of the unfolded protein response (UPR), can confer beneficial effects by facilitating the removal of cytosolic aggregates through the autophagy-lysosome pathway (ALP). In neurodegenerative diseases, the ALP ameliorates the accumulation of intracellular protein aggregates in the brain. Transcription factor-EB (TFEB), a master regulator of the ALP, positively regulates key genes involved in the cellular degradative pathway. However, in neurons, the role of PERK activation in mitigating amyloidogenesis by ALP remains unclear. In this study, we found that SB202190 selectively activates PERK independently of its inhibition of p38 mitogen-activated protein kinase, but not inositol-requiring transmembrane kinase/endoribonuclease-1α (IRE1α) or activating transcription factor 6 (ATF6), in human neuroblastoma cells. PERK activation by SB202190 was dependent on mitochondrial ROS production and promoted Ca2+-calcineurin activation. The activation of the PERK-Ca2+-calcineurin axis by SB202190 positively affects TFEB activity to increase ALP in neuroblastoma cells. Collectively, our study reveals a novel physiological mechanism underlying ALP activation, dependent on PERK activation, for ameliorating amyloidogenesis in neurodegenerative diseases.

Anti-4-1BB antibody-based combination therapy augments antitumor immunity by enhancing CD11c+CD8+ T cells in renal cell carcinoma.

To improve the potential treatment strategies of incurable renal cell carcinoma (RCC), which is highly resistant to chemotherapy and radiotherapy, the present study established a combination therapy with immunostimulatory factor (ISTF) and anti-4-1BB monoclonal antibodies (mAbs) to augment the antitumor response in a murine RCC model. ISTF isolated from Actinobacillus actinomycetemcomitans stimulates macrophages, dendritic cells and B cells to produce IL-6, TNF-α, nitric oxide and major histocompatibility complex class II expression. 4-1BB (CD137) is expressed in activated immune cells, including activated T cells, and is a promising target for cancer immunotherapy. The administration of anti-4-1BB mAbs promoted antitumor immunity via enhancing CD11c+CD8+ T cells. The CD11c+CD8+ T cells were characterized by high killing activity and IFN-γ-producing ability, representing a phenotype of active effector cytotoxic T lymphocytes. The present study showed that combination therapy with ISTF and anti-4-1BB mAbs promoted partial tumor regression with established RCC, but monotherapy with ISTF or anti-4-1BB mAbs did not. These effects were speculated to be caused by the increase in CD11c+CD8+ T cells in the spleen and tumor, and IFN-γ production. These insights into the effector mechanisms of the combination of ISTF and anti-4-1BB mAbs may be useful for targeting incurable RCC.

Potential Role of Heme Oxygenase-1 in the Resolution of Experimentally Induced Colitis through Regulation of Macrophage Polarization.

BACKGROUND/AIMS: Heme oxygenase-1 (HO-1) plays a central role in cellular defense against inflammatory insults, and its induction in macrophages potentiates their efferocytic activity. In this study, we explored the potential role of macrophage HO-1 in the resolution of experimentally induced colitis. METHODS: To induce colitis, male C57BL/6 mice were treated with 2% dextran sulfate sodium (DSS) in the drinking water for 7 days. To investigate efferocytosis, apoptotic colon epithelial CCD 841 CoN cells were coincubated with bone marrow-derived macrophages (BMDMs). RESULTS: Administration of the HO-1 inhibitor zinc protoporphyrin IX (ZnPP) blunted the resolution of DSS-induced intestinal inflammation and expression of the proresolving M2 macrophage marker CD206. BMDMs treated with apoptotic colonic epithelial cells showed significantly elevated expression of HO-1 and its regulator Nrf2. Under the same experimental conditions, the proportion of CD206-expressing macrophages was also enhanced. ZnPP treatment abrogated the upregulation of CD206 expression in BMDMs engulfing apoptotic colonic epithelial cells. This result was verified with BMDMs isolated from HO-1-knockout mice. BMDMs, when stimulated with lipopolysaccharide, exhibited increased expression of CD86, a marker of M1 macrophages. Coculture of lipopolysaccharide-stimulated BMDMs with apoptotic colonic epithelial cell debris dampened the expression of CD86 as well as the pro-inflammatory cytokines in an HO-1-dependent manner. Genetic ablation as well as pharmacologic inhibition of HO-1 significantly reduced the proportion of efferocytic BMDMs expressing the scavenger receptor CD36. CONCLUSIONS: HO-1 plays a key role in the resolution of experimentally induced colitis by modulating the polarization of macrophages.

Reprograming of Tumor-Associated Macrophages in Breast Tumor-Bearing Mice under Chemotherapy by Targeting Heme Oxygenase-1.

Tumor-associated macrophages (TAMs) represent one of the most abundant components of the tumor microenvironment and play important roles in tumor development and progression. TAMs display plasticity and functional heterogeneity as reflected by distinct phenotypic subsets. TAMs with an M1 phenotype have proinflammatory and anti-tumoral properties whereas M2-like TAMs exert anti-inflammatory and pro-tumoral functions. Tumor cell debris generated during chemotherapy can stimulate primary tumor growth and recurrence. According to our previous study, phagocytic engulfment of breast tumor cell debris by TAMs attenuated chemotherapeutic efficacy through the upregulation of heme oxygenase-1 (HO-1). To verify the impact of HO-1 upregulation on the profile of macrophage polarization during cytotoxic therapy, we utilized a syngeneic murine breast cancer (4T1) model in which tumor bearing mice were treated with paclitaxel (PTX). PTX treatment markedly downregulated the surface expression of the M1 marker CD86 in infiltrated TAMs. Notably, there were significantly more cytotoxic CD8+ T cells in tumors of mice treated with PTX plus the HO-1 inhibitor, zinc protophorphyrin IX (ZnPP) than in mice treated with PTX alone. Interestingly, the tumor-inhibiting efficacy of PTX and ZnPP co-treatment was abrogated when macrophages were depleted by clodronate liposomes. Macrophage depletion also decreased the intratumoral CD8+ T cell population and downregulated the expression of Cxcl9 and Cxcl10. The expression of the M1 phenotype marker, CD86 was higher in mice injected with PTX plus ZnPP than that in mice treated with PTX alone. Conversely, the PTX-induced upregulation of the M2 marker gene, Il10 in CD11b+ myeloid cells from 4T1 tumor-bearing mice treated was dramatically reduced by the administration of the HO-1 inhibitor. Genetic ablation of HO-1 abolished the inhibitory effect of 4T1 tumor cell debris on expression of M1 marker genes, Tnf and Il12b, in LPS-stimulated BMDMs. HO-1-deficient BMDMs exposed to tumor cell debris also exhibited a diminished expression of the M2 macrophage marker, CD206. These findings, taken all together, provide strong evidence that HO-1 plays a pivotal role in the transition of tumor-inhibiting M1-like TAMs to tumor-promoting M2-like ones during chemotherapy.

Breast cancer cell debris diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor-associated macrophages.

Chemotherapy is commonly used as a major therapeutic option for breast cancer treatment, but its efficacy is often diminished by disruption of patient's anti-tumor immunity. Chemotherapy-generated tumor cell debris could hijack accumulated tumor-associated macrophages (TAMs), provoking tumor recurrence. Therefore, reprogramming TAMs to acquire an immunocompetent phenotype is a promising strategy to potentiate therapeutic efficacy. In this study, we analyzed the proportion of immune cells in the breast cancer patients who received chemotherapy. To validate our findings in vivo, we used a syngeneic murine breast cancer (4T1) model. Chemotherapy generates an immunosuppressive tumor microenvironment in breast cancer. Here, we show that phagocytic engulfment of tumor cell debris by TAMs reduces chemotherapeutic efficacy in a 4T1 breast cancer model. Specifically, the engulfment of tumor cell debris by macrophages reduced M1-like polarization through heme oxygenase-1 (HO-1) upregulation. Conversely, genetic or pharmacologic inhibition of HO-1 in TAMs restored the M1-like polarization. Our results demonstrate that tumor cell debris-induced HO-1 expression in macrophages regulates their polarization. Inhibition of HO-1 overexpression in TAMs may provoke a robust anti-tumor immune response, thereby potentiating the efficacy of chemotherapy.

GSK-3ß inhibition by curcumin mitigates amyloidogenesis via TFEB activation and anti-oxidative activity in human neuroblastoma cells.

The translocation of transcription factor EB (TFEB) to the nucleus plays a pivotal role in the regulation of basic cellular processes, such as lysosome biogenesis and autophagy. Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome, which is important in maintaining cellular homeostasis during environmental stress. Furthermore, oxidative stress is a critical cause for the progression of neurodegenerative diseases. Curcumin has anti-oxidative and anti-inflammatory activities, and is expected to have potential therapeutic effects in various diseases. In this study, we demonstrated that curcumin regulated TFEB export signalling via inhibition of glycogen synthase kinase-3ß (GSK-3ß); GSK-3ß was inactivated by curcumin, leading to reduced phosphorylation of TFEB. We further showed that H2O2-induced oxidative stress was reduced by curcumin via the Nrf2/HO-1 pathway in human neuroblastoma cells. In addition, we showed that curcumin induced the degradation of amyloidogenic proteins, including amyloid-ß precursor protein and α-synuclein, through the TFEB-autophagy/lysosomal pathway. In conclusion, curcumin regulates autophagy by controlling TFEB through the inhibition of GSK-3ß, and increases antioxidant gene expression in human neuroblastoma cells. These results contribute to the development of novel cellular therapies for neurodegenerative diseases.

Interleukin-8 drives CD38 to form NAADP from NADP+ and NAAD in the endolysosomes to mobilize Ca2+ and effect cell migration.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger whose formation has remained elusive. In vitro, CD38-mediated NAADP synthesis requires an acidic pH and a nonphysiological concentration of nicotinic acid (NA). We discovered that CD38 catalyzes synthesis of NAADP by exchanging the nicotinamide moiety of nicotinamide adenine dinucleotide phosphate (NADP+ ) for the NA group of nicotinic acid adenine dinucleotide (NAAD) inside endolysosomes of interleukin 8 (IL8)-treated lymphokine-activated killer (LAK) cells. Upon IL8 stimulation, cytosolic NADP+ is transported to acidified endolysosomes via connexin 43 (Cx43) and gated by cAMP-EPAC-RAP1-PP2A signaling. CD38 then performs a base-exchange reaction with the donor NA group deriving from NAAD, produced by newly described endolysosomal activities of NA phosphoribosyltransferase (NAPRT) and NMN adenyltransferase (NMNAT) 3. Thus, the membrane organization of endolysosomal CD38, a signal-mediated transport system for NADP+ and luminal NAD+ biosynthetic enzymes integrate signals from a chemokine and cAMP to specify the spatiotemporal mobilization of Ca2+ to drive cell migration.

Cross-talk between CD38 and TTP Is Essential for Resolution of Inflammation during Microbial Sepsis.

The resolution phase of acute inflammation is essential for tissue homeostasis, yet the underlying mechanisms remain unclear. We demonstrate that resolution of inflammation involves interactions between CD38 and tristetraprolin (TTP). During the onset of acute inflammation, CD38 levels are increased, leading to the production of Ca2+-signaling messengers, nicotinic acid adenine dinucleotide phosphate (NAADP), ADP ribose (ADPR), and cyclic ADPR (cADPR) from NAD(P)+. To initiate the onset of resolution, TTP expression is increased by the second messengers, NAADP and cADPR, which downregulate CD38 expression. The activation of TTP by Sirt1-dependent deacetylation, in response to increased NAD+ levels, suppresses the acute inflammatory response and decreases Rheb expression, inhibits mTORC1, and induces autophagolysosomes for bacterial clearance. TTP may represent a mechanistic target of anti-inflammatory agents, such as carbon monoxide. TTP mediates crosstalk between acute inflammation and autophagic clearance of bacteria from damaged tissue in the resolution of inflammation during sepsis.

Carbon monoxide ameliorates acetaminophen-induced liver injury by increasing hepatic HO-1 and Parkin expression.

Acetaminophen (APAP) is widely used as an antifebrile and analgesic drug at recommended doses, whereas an overdose of APAP can cause severe liver damage. The molecular mechanisms underlying APAP-induced liver damage remain incompletely understood. Carbon monoxide (CO), an end-product of heme oxygenase (HO)-1 activity, can confer anti-inflammatory and antiapoptotic properties in cellular models of toxicity via regulation of mitochondrial function. The objective of this study was to evaluate the effects of CO on APAP-induced hepatotoxicity and CO's relationship to regulation of endoplasmic reticulum (ER) stress and mitochondrial signaling using CO-releasing molecules or low concentrations of CO applied as pretreatment or posttreatment. Using genetic deletion or knockdown approaches in alpha mouse liver cells or primary hepatocytes, respectively, we investigated the role of HO-1 and the mitophagy regulator protein Parkin on APAP-induced expression of the ER stress-associated apoptosis regulator cytosine-cytosine-adenosine-adenosine-thymidine (CCAAT)/enhancer-binding protein homologous protein (CHOP). We found that CO induced Parkin expression in hepatocytes via the protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2-α/activating transcription factor-4 signaling pathway. Additionally, CO gas inhalation significantly alleviated APAP-induced liver damage in vivo and correspondingly reduced serum alanine aminotransferase and aspartate aminotransferase levels as well as proinflammatory cytokines and reduced the expression of CHOP in liver tissues while dramatically increasing hepatic HO-1 and Parkin expression. We found that the protective effects of CO on APAP-induced liver damage were mediated by down-regulation of CHOP at a transcriptional and post-translational level via induction of HO-1 and Parkin, respectively, and associated with decreases in reactive oxygen species production and JNK phosphorylation. We conclude that CO may represent a promising therapeutic agent for APAP-induced liver injury.-Chen, Y., Park, H.-J., Park, J., Song, H.-C., Ryter, S. W., Surh, Y.-J., Kim, U.-H., Joe, Y., Chung, H. T. Carbon monoxide ameliorates acetaminophen-induced liver injury by increasing hepatic HO-1 and Parkin expression.

Similarities and Distinctions in the Effects of Metformin and Carbon Monoxide in Immunometabolism.

Immunometabolism, defined as the interaction of metabolic pathways with the immune system, influences the pathogenesis of metabolic diseases. Metformin and carbon monoxide (CO) are two pharmacological agents known to ameliorate metabolic disorders. There are notable similarities and differences in the reported effects of metformin and CO on immunometabolism. Metformin, an anti-diabetes drug, has positive effects on metabolism and can exert anti-inflammatory and anti-cancer effects via adenosine monophosphate-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms. CO, an endogenous product of heme oxygenase-1 (HO-1), can exert anti-inflammatory and antioxidant effects at low concentration. CO can confer cytoprotection in metabolic disorders and cancer via selective activation of the protein kinase R-like endoplasmic reticulum (ER) kinase (PERK) pathway. Both metformin and CO can induce mitochondrial stress to produce a mild elevation of mitochondrial ROS (mtROS) by distinct mechanisms. Metformin inhibits complex I of the mitochondrial electron transport chain (ETC), while CO inhibits ETC complex IV. Both metformin and CO can differentially induce several protein factors, including fibroblast growth factor 21 (FGF21) and sestrin2 (SESN2), which maintain metabolic homeostasis; nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of the antioxidant response; and REDD1, which exhibits an anticancer effect. However, metformin and CO regulate these effects via different pathways. Metformin stimulates p53- and AMPK-dependent pathways whereas CO can selectively trigger the PERK-dependent signaling pathway. Although further studies are needed to identify the mechanistic differences between metformin and CO, pharmacological application of these agents may represent useful strategies to ameliorate metabolic diseases associated with altered immunometabolism.

Carbon monoxide attenuates amyloidogenesis via down-regulation of NF-κB-mediated BACE1 gene expression.

Amyloid-ß (Aß) peptides, the major constituent of plaques, are generated by sequential proteolytic cleavage of the amyloid precursor protein (APP) via ß-secretase (BACE1) and the γ-secretase complex. It has been proposed that the abnormal secretion and accumulation of Aß are the initial causative events in the development of Alzheimer's disease (AD). Drugs modulating this pathway could be used for AD treatment. Previous studies indicated that carbon monoxide (CO), a product of heme oxygenase (HO)-1, protects against Aß-induced toxicity and promotes neuroprotection. However, the mechanism underlying the mitigative effect of CO on Aß levels and BACE1 expression is unclear. Here, we show that CO modulates cleavage of APP and Aß production by decreasing BACE1 expression in vivo and in vitro. CO reduces Aß levels and improves memory deficits in AD transgenic mice. The regulation of BACE1 expression by CO is dependent on nuclear factor-kappa B (NF-κB). Consistent with the negative role of SIRT1 in the NF-κB activity, CO fails to evoke significant decrease in BACE1 expression in the presence of the SIRT1 inhibitor. Furthermore, CO attenuates elevation of BACE1 level in brains of 3xTg-AD mouse model as well as mice fed high-fat, high-cholesterol diets. CO reduces the NF-κB-mediated transcription of BACE1 induced by the cholesterol oxidation product 27-hydroxycholesterol or hydrogen peroxide. These data suggest that CO reduces the NF-κB-mediated BACE1 transcription and consequently decreases Aß production. Our study provides novel mechanisms by which CO reduces BACE1 expression and Aß production and may be an effective agent for AD treatment.

The Essential Role of Ca2+ Signals in UVB-Induced IL-1ß Secretion in Keratinocytes.

UVB-induced skin damage is attributable to reactive oxygen species, which are triggered by intracellular Ca2+ signals. However, exactly how the reactive oxygen species are triggered by intracellular Ca2+ upon UVB irradiation remains obscure. Here, we show that UVB induces Ca2+ signals via sequential generation of the following Ca2+ messengers: inositol 1,4,5-trisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose. UVB induced H2O2 production through NADPH oxidase 4 activation, which is downstream to inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate. H2O2 derived from NADPH oxidase 4 activated CD38 to produce cyclic ADP-ribose. UVB first evoked the pannexin channel to release ATP, which acts on P2X7 receptor to generate inositol 1,4,5-trisphosphate. Inhibitors of these messengers, as well as antioxidants, blocked UVB-induced Ca2+ signals and IL-1ß secretion in keratinocytes. Furthermore, ablation of CD38 and NADPH oxidase 4 protected against UVB-induced inflammation and IL-1ß secretion in the murine epidermis. These results show that UVB induces IL-1ß secretion through cross-talk between Ca2+ and reactive oxygen species, providing insight towards potential targets against UVB-induced inflammation.

Pterostilbene 4'-ß-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1.

Heme oxygenase-1 (HO-1) can exert anti-inflammatory and antioxidant effects. Acute lung injury (ALI) is associated with increased inflammation and influx of proinflammatory cells and mediators in the airspaces and lung parenchyma. In this study, we demonstrate that pterostilbene 4'-ß-glucoside (4-PG), the glycosylated form of the antioxidant pterostilbene (PTER), can protect against lipopolysaccharide- (LPS-) or Pseudomonas aeruginosa- (P. aeruginosa-) induced ALI when applied as a pretreatment or therapeutic post-treatment, via the induction of HO-1. To determine whether HO-1 mediates the antioxidant and anti-inflammatory effects of 4-PG, we subjected mice genetically deficient in Hmox-1 to LPS-induced ALI and evaluated histological changes, HO-1 expression, and proinflammatory cytokine levels in bronchoalveolar lavage (BAL) fluid. 4-PG exhibited protective effects on LPS- or P. aeruginosa-induced ALI by ameliorating pathological changes in lung tissue and decreasing proinflammatory cytokines. In addition, HO-1 expression was significantly increased by 4-PG in cells and in mouse lung tissues. The glycosylated form of pterostilbene (4-PG) was more effective than PTER in inducing HO-1 expression. Genetic deletion of Hmox-1 abolished the protective effects of 4-PG against LPS-induced inflammatory responses. Furthermore, we found that 4-PG decreased both intracellular ROS levels and mitochondrial (mt) ROS production in a manner dependent on HO-1. Pharmacological application of the HO-1 reaction product carbon monoxide (CO), but not biliverdin or iron, conferred protection in Hmox-1-deficient macrophages. Taken together, these results demonstrate that 4-PG can increase HO-1 expression, which plays a critical role in ameliorating intracellular and mitochondrial ROS production, as well as in downregulating inflammatory responses induced by LPS. Therefore, these findings strongly suggest that HO-1 mediates the antioxidant and anti-inflammatory effects of 4-PG.

Quercetin Reduces Tumor Necrosis Factor Alpha-Induced Muscle Atrophy by Upregulation of Heme Oxygenase-1.

The inflammatory cytokine tumor necrosis factor α (TNFα), upregulated in the obese condition, promotes protein degradation and is implicated in obesity-related skeletal muscle atrophy and age-related sarcopenia. Quercetin, a flavonoid, elicits antioxidative and anti-inflammatory activities. In this study, we investigated the effect of quercetin on TNFα-induced skeletal muscle atrophy as well as its potential mechanism of action. In this study, we observed that quercetin suppressed expression of TNFα-induced atrophic factors such as MAFbx/atrogin-1 and MuRF1 in myotubes, and it enhanced heme oxygenase-1 (HO-1) protein level accompanied by increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in myotubes. The HO-1 inhibitor ZnPP suppressed the inhibitory actions of quercetin on TNFα-induced atrophic responses and degradation of IκB-α in myotubes. Moreover, quercetin supplementation to high-fat diet-fed obese mice inhibited obesity-induced atrophic responses in skeletal muscle, accompanied by upregulation of HO-1 and inactivation of nuclear factor-kappa B (NF-κB), and the quercetin actions were attenuated in Nrf2-deficient mice. These findings suggest that quercetin protects against TNFα-induced muscle atrophy under obese conditions through Nrf2-mediated HO-1 induction accompanied by inactivation of NF-κB. Quercetin may be used as a dietary supplement to protect against obesity-induced skeletal muscle atrophy.

Role of heme oxygenase-1 in potentiation of phagocytic activity of macrophages by taurine chloramine: Implications for the resolution of zymosan A-induced murine peritonitis.

Phagocytosis of pathogens by macrophages is crucial for the successful resolution of inflammation induced by microbial infection. Taurine chloramine (TauCl), an endogenous anti-inflammatory and antioxidative substance, is produced by reaction between taurine and hypochlorous acid by myeloperoxidase activity in neutrophils under inflammatory conditions. In the present study, we investigated the effect of TauCl on resolution of acute inflammation caused by fungal infection using a zymosan A-induced murine peritonitis model. TauCl administration reduced the number of the total peritoneal leukocytes, while it increased the number of peritoneal monocytes. Furthermore, TauCl promoted clearance of pathogens remaining in the inflammatory environment by macrophages. When the macrophages isolated from thioglycollate-treated mice were treated with TauCl, their phagocytic capability was enhanced. In the murine macrophage-like RAW264.7 cells treated with TauCl, the proportion of macrophages clearing the zymosan A particles was also increased. TauCl administration resulted in elevated expression of heme oxygenase-1 (HO-1) in the peritoneal macrophages. Pharmacologic inhibition of HO-1 activity or knockdown of HO-1 in the murine macrophage RAW264.7 cells abolished the TauCl-induced phagocytosis, whereas the overexpression of HO-1 augmented the phagocytic ability of macrophages. Moreover, peritoneal macrophages isolated from HO-1 null mice failed to mediate TauCl-induced phagocytosis. Our results suggest that TauCl potentiates phagocytic activity of macrophages through upregulation of HO-1 expression.

FGF21 induced by carbon monoxide mediates metabolic homeostasis via the PERK/ATF4 pathway.

The prevalence of metabolic diseases, including type 2 diabetes, obesity, and cardiovascular disease, has rapidly increased, yet the molecular mechanisms underlying the metabolic syndrome, a primary risk factor, remain incompletely understood. The small, gaseous molecule carbon monoxide (CO) has well-known anti-inflammatory, antiproliferative, and antiapoptotic effects in a variety of cellular- and tissue-injury models, whereas its potential effects on the complex pathways of metabolic disease remain unknown. We demonstrate here that CO can alleviate metabolic dysfunction in vivo and in vitro. We show that CO increased the expression and section of the fibroblast growth factor 21 (FGF21) in hepatocytes and liver. CO-stimulated PERK activation and enhanced the levels of FGF21 via the eIF2α-ATF4 signaling pathway. The induction of FGF21 by CO attenuated endoreticulum stress- or diet-induced, obesity-dependent hepatic steatosis. Moreover, CO inhalation lowered blood glucose levels, enhanced insulin sensitivity, and promoted energy expenditure by stimulating the emergence of beige adipose cells from white adipose cells. In conclusion, we suggest that CO acts as a potent inducer of FGF21 expression and that CO critically depends on FGF21 to regulate metabolic homeostasis.-Joe, Y., Kim, S., Kim, H. J., Park, J., Chen, Y., Park, H.-J., Jekal, S.-J., Ryter, S. W., Kim, U. H., Chung, H. T. FGF21 induced by carbon monoxide mediates metabolic homeostasis via the PERK/ATF4 pathway.

The novel resveratrol derivative 3,5-diethoxy-3',4'-dihydroxy-trans-stilbene induces mitochondrial ROS-mediated ER stress and cell death in human hepatoma cells in vitro.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a well-known polyphenol that is present in grapes, peanuts, pine seeds, and several other plants. Resveratrol exerts deleterious effects on various types of human cancer cells. Here, we analyzed the cell death-inducing mechanisms of resveratrol-006 (Res-006), a novel resveratrol derivative in human liver cancer cells in vitro. Res-006 was more effectively suppressed the viability of HepG2 human hepatoma cells than resveratrol (the IC50 values were 67.2 and 354.8 µmol/L, respectively). Co-treatment with the ER stress regulator 4-phenylbutyrate (0.5 mmol/L) or the ROS inhibitor N-acetyl-L-cysteine (NAC, 1 mmol/L) significantly attenuated Res-006-induced HepG2 cell death, suggesting that pro-apoptotic ER stress and/or ROS may govern the Res-006-induced HepG2 cell death. We further revealed that treatment of HepG2 cells with Res-006 (65 µmol/L) immediately elicited the dysregulation of mitochondrial dynamics and the accumulation of mitochondrial ROS. It also collapsed the mitochondrial membrane potential and further induced ER stress and cell death. These events, except for the change in mitochondrial morphology, were prevented by the exposure of the HepG2 cells to the mitochondrial ROS scavenger, Mito-TEMPO (300-1000 µmol/L). The results suggest that Res-006 may kill HepG2 cells through cell death pathways, including the ER stress initiated by mitochondrial ROS accumulation. The cell death induced by this novel resveratrol derivative involves crosstalk between the mitochondria and ER stress mechanisms.