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.

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.

Carbon monoxide induces the assembly of stress granule through the integrated stress response.

Stress granules (SGs) are membraneless and phase-dense organelles that form transiently in response to a variety of harmful stimuli, including oxidative, heat, osmotic, ultraviolet light and chemotoxic stresses, and thus providing protective effects, allowing survivals. Carbon monoxide (CO), a gaseous second messenger, is synthesized by heme-oxygenases, and exerts anti-inflammatory, anti-proliferative and anti-apoptotic effects in a variety of cellular- and tissue-injury models. Several reports indicate that low levels of mitochondrial reactive oxygen species (mtROS) generated by CO can selectively activate PERK-eIF2α integrated stress response (ISR) to preserve the cellular homeostasis. Hence, CO can confer protection against cellular stresses. However, the mechanisms underlying the cyto-protective effects of CO against various harmful stimuli remain to be elucidated. Here, we sought to examine whether CO induces the SG assembly, and uncover its molecular mechanisms. We treated WI-38 cells and primary mouse embryonic fibroblasts (MEFs) with CO-releasing molecule 2 (CORM2) or CO gas, and found the SG assemblies were gradually increased in time and dose dependent manners. Next, we used Mito-TEMPO, an mtROS scavenger, to explore if mtROS might be involved in the CO-induced SG assembly. Furthermore, we confirmed the involvement of ISR consisted of PERK-eIF2α signaling pathway induced by CO for the SGs assembly. Finally, the inhibition of SG assembly by ISR inhibitor further verified CO-induced ISR might be responsible for SG. Taken together, in this study, we first demonstrated that CO is a novel SG inducer by activating ISR. Moreover, mtROS might be an initiator for the CO-induced ISR responsible for SG assembly.

CO ameliorates endothelial senescence induced by 5-fluorouracil through SIRT1 activation.

Endothelial senescence is the main risk factor that contributes to vascular dysfunction and the progression of vascular disease. Carbon monoxide (CO) plays an important role in preventing vascular dysfunction and in maintaining vascular physiology or homeostasis. The application of exogenous CO has been shown to confer protection in several models of cardiovascular injury or disease, including hypertension, atherosclerosis, balloon-catheter injury, and graft rejection. However, the mechanism by which CO prevents endothelial senescence has been largely unexplored. The aim of this study was to evaluate the effects of CO on endothelial senescence and to investigate the possible mechanisms underlying this process. We measured the levels of senescence-associated-ß-galactosidase activity, senescence-associated secretory phenotype, reactive oxygen species (ROS) production, and stress granule in human umbilical vein endothelial cells and the WI-38 human diploid fibroblast cell line. We found that 5-fluorouracil (5FU)-induced ROS generation was inhibited by CO-releasing molecules (CORM)-A1 treatment, and endothelial senescence induced by 5FU was attenuated by CORM-A1 treatment. The SIRT1 inhibitor EX527 reversed the inhibitory effect of CO on the 5FU-induced endothelial senescence. Furthermore, SIRT1 deficiency abolished the stress granule formation by CO. Our results suggest that CO alleviates the endothelial senescence induced by 5FU through SIRT1 activation and may hence have therapeutic potential for the treatment of vascular diseases.

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.

Taurine chloramine potentiates phagocytic activity of peritoneal macrophages through up-regulation of dectin-1 mediated by heme oxygenase-1-derived carbon monoxide.

Resolution of inflammation that occurs after microbial infection or tissue damage is an important physiologic process in maintaining or restoring host homeostasis. Taurine chloramine (TauCl) is formed by a reaction between taurine and hypochlorite in leukocytes, and it is especially abundant in activated neutrophils that encounter an oxidative burst. As neutrophils undergo apoptosis, TauCl is released to the extracellular matrix at the inflamed sites, thereby affecting coexisting macrophages in the inflammatory microenvironment. In this study, we investigated the role of TauCl in phagocytosis by macrophages during resolution of fungal infection-induced inflammation. We found that exogenous TauCl substantially increased the phagocytic efficiency of macrophages through up-regulation of dectin-1, a receptor for fungal ß-1,3-glucans, which is present on the membrane of macrophages. Our previous studies demonstrated the induction of heme oxygenase-1 (HO-1) expression in murine peritoneal macrophages treated with TauCl. In the present study, knocking out HO-1 or pharmacologic inhibition of HO-1 with zinc protoporphyrin IX attenuated the TauCl-induced expression of dectin-1 and subsequent phagocytosis. Furthermore, carbon monoxide (CO), a by-product of the HO-1-catalyzed reaction, induced expression of dectin-1 and potentiated phagocytic capability of the macrophages, which appeared to be mediated through up-regulation of peroxisome proliferator-activated receptor γ. Taken together, induction of HO-1 expression and subsequent CO production by TauCl are essential for phagocytosis of fungi by macrophages. Our results suggest that TauCl has important roles in host defense against fungal infection and has therapeutic potential in the management of inflammatory diseases.-Kim, S. H., Zhong, X., Kim, W., Kim, K., Suh, Y.-G., Kim, C., Joe, Y., Chung, H. T., Cha, Y.-N., Surh, Y.-J. Taurine chloramine potentiates phagocytic activity of peritoneal macrophages through up-regulation of dectin-1 mediated by heme oxygenase-1-derived carbon monoxide.

Fibroblast growth factor 21 deficiency aggravates obesity-induced hypothalamic inflammation and impairs thermogenic response.

OBJECTIVE AND DESIGN: Hypothalamic inflammation is closely associated with metabolic dysregulation. Fibroblast growth factor 21 (FGF21) is known to be an important metabolic regulator with anti-inflammatory properties. In this study, we investigated the effects of FGF21 deficiency on obesity-induced hypothalamic inflammation and thermogenic responses. MATERIALS AND METHODS: FGF21-deficient mice and/or wild-type (WT) mice were fed a high-fat diet (HFD) for 12 weeks. RESULTS: FGF21-deficient mice fed an HFD showed increased levels of inflammatory cytokines compared with WT obese control, and this was accompanied by upregulation of gliosis markers in the hypothalamus. Expression of heat-shock protein 72, a marker of neuronal damage, was increased in the FGF21-deficient obese mice, and the expression of hypothalamic neuronal markers involved in anti-thermogenic or thermogenic responses was altered. Moreover, the protein level of uncoupling protein 1 and other thermogenic genes were markedly reduced in the brown adipose tissue of the FGF21-deficient obese mice. CONCLUSIONS: These findings suggest that FGF21 deficiency aggravates obesity-induced hypothalamic inflammation and neuronal injury, leading to alterations in hypothalamic neural circuits accompanied by a reduction of the thermogenic response.

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.

Both nitric oxide and nitrite prevent homocysteine-induced endoplasmic reticulum stress and subsequent apoptosis via cGMP-dependent pathway in neuronal cells.

Growing evidence indicates that endoplasmic reticulum (ER) stress and/or ER stress-mediated apoptosis may play a role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. The present study investigated the effects of non-cytotoxic concentrations of nitric oxide (NO) and nitrite, a metabolite of NO, on ER stress and ER stress-mediated apoptosis in Neuro-2a cells exposed to homocysteine (Hcy), an endogenous ER stress inducer. Hcy induced ER stress, as confirmed by inositol-requiring enzyme 1α (IRE1α) phosphorylation and X-box-binding protein-1 (Xbp1) mRNA splicing as well as C/EBP homologous protein (CHOP) expression, and apoptosis, as verified by Annexin V-positive cells. Surprisingly, non-cytotoxic NO (S-nitrosoglutathione) and nitrite markedly reduced Hcy-induced IRE1α phosphorylation, Xbp1 mRNA splicing, CHOP expression, and Annexin V-positive cells, indicating the cytoprotection of NO and nitrite against Hcy-induced ER stress and apoptosis. Moreover, inhibition of sGC/cGMP pathway abolished the cytoprotective effects of NO and nitrite, whereas cellular elevation of cGMP levels mimicked the cytoprotective actions of NO and nitrite. These findings provide the first evidence showing that both NO and nitrite can reduce ER stress and subsequent apoptosis via NO-sGC-cGMP pathway in neuronal cells and suggesting that NO and/or nitrite may have therapeutic value in the treatment of ER stress-associated neurodegenerative diseases.

Piceatannol attenuates homocysteine-induced endoplasmic reticulum stress and endothelial cell damage via heme oxygenase-1 expression.

A growing body of evidence implicates endoplasmic reticulum (ER)-induced cellular dysfunction and apoptosis as important factors to a variety of diseases. In endothelial cells (ECs), the sulfur-containing amino acid homocysteine (Hcy) causes EC apoptosis and reactive oxygen species (ROS) generation through induction of ER stress. Here, we have investigated whether piceatannol (Pic), a resveratrol analog, could protect ECs against Hcy-induced apoptosis, oxidative stress and ER stress, with specific emphasis on heme oxygenase-1 (HO-1). In human ECs, we determined the effects of Hcy and Pic on annexin V positivity, glucose-regulated protein 78 kDa (GRP78) and C/EBP homologous protein (CHOP) expression, X-box binding protein 1 (Xbp-1) mRNA slicing, and ROS-sensitive dihydroethidium (DHE) oxidation. Hcy increased annexin V-positive cells, DHE oxidation, GRP78 and CHOP expression and Xbp-1 mRNA splicing, indicating that Hcy induces apoptosis, oxidative stress and ER stress. Pretreatment of ECs with Pic significantly inhibited Hcy-induced apoptosis, ROS generation and ER stress. Pic also increased HO-1 expression via activation of nuclear factor-E2-related factor 2 (Nrf2). Interestingly, the inhibitory effects of Pic on Hcy-induced apoptosis, ROS generation and ER stress were abolished by down-regulation of HO-1 expression, while mimicked by treatment of ECs with the HO-1 inducer hemin. Overall, these results suggest that Pic may protect ECs against Hcy-induced apoptosis, oxidative stress and ER stress via Nrf2-dependent HO-1 expression.

Endoplasmic reticulum stress-induced IRE1α activation mediates cross-talk of GSK-3ß and XBP-1 to regulate inflammatory cytokine production.

IL-1ß and TNF-α are important proinflammatory cytokines that respond to mutated self-antigens of tissue damage and exogenous pathogens. The endoplasmic reticulum (ER) stress and unfolded protein responses are related to the induction of proinflammatory cytokines. However, the detailed molecular pathways by which ER stress mediates cytokine gene expression have not been investigated. In this study, we found that ER stress-induced inositol-requiring enzyme (IRE)1α activation differentially regulates proinflammatory cytokine gene expression via activation of glycogen synthase kinase (GSK)-3ß and X-box binding protein (XBP)-1. Surprisingly, IL-1ß gene expression was modulated by IRE1α-mediated GSK-3ß activation, but not by XBP-1. However, IRE1α-mediated XBP-1 splicing regulated TNF-α gene expression. SB216763, a GSK-3 inhibitor, selectively inhibited IL-1ß gene expression, whereas the IRE1α RNase inhibitor STF083010 suppressed only TNF-α production. Additionally, inhibition of GSK-3ß greatly increased IRE1α-dependent XBP-1 splicing. Our results identify an unsuspected differential role of downstream mediators GSK-3ß and XBP-1 in ER stress-induced IRE1α activation that regulates cytokine production through signaling cross-talk. These results have important implications in the regulation of inflammatory pathways during ER stress, and they suggest novel therapeutic targets for diseases in which meta-inflammation plays a key role.

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.

Carbon monoxide protects against hepatic ischemia/reperfusion injury by modulating the miR-34a/SIRT1 pathway.

Hepatic ischemia/reperfusion (I/R) injury can arise as a complication of liver surgery and transplantation. Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, modulates inflammation and apoptosis in response to oxidative stress. SIRT1, which is regulated by p53 and microRNA-34a (miR-34a), can modulate non-alcoholic fatty liver disease, fibrosis and cirrhosis. Since carbon monoxide (CO) inhalation can protect against hepatic I/R, we hypothesized that CO could ameliorate hepatic I/R injury by regulating the miR-34a/SIRT1 pathway. Livers from mice pretreated with CO, or PFT, a p53 inhibitor, displayed reduced production of pro-inflammatory mediators, including TNF-α, iNOS, interleukin (IL)-6, and IL-1ß after hepatic I/R injury. SIRT1 expression was increased by CO or PFT in the liver after I/R, whereas acetylated p65, p53 levels, and miR-34a expression were decreased. CO increased SIRT1 expression by inhibiting miR-34a. Both CO and PFT diminished pro-inflammatory cytokines production in vitro. Knockdown of SIRT1 in LPS-stimulated macrophages increased NF-κB acetylation, and increased pro-inflammatory cytokines. CO treatment reduced miR-34a expression and increased SIRT1 expression in oxidant-challenged hepatocytes; and rescued SIRT1 expression in p53-expressing or miR-34a transfected cells. In response to CO, enhanced SIRT1 expression mediated by miR-34a inhibition protects against liver damage through p65/p53 deacetylation, which may mediate inflammatory responses and hepatocellular apoptosis. The miR-34a/SIRT1 pathway may represent a therapeutic target for hepatic injury.

Tristetraprolin mediates the anti-proliferative effects of metformin in breast cancer cells.

Metformin, which is a drug commonly prescribed to treat type 2 diabetes, has anti-proliferative effects in cancer cells; however, the molecular mechanisms underlying this effect remain largely unknown. The aim is to investigate the role of tristetraprolin (TTP), an AU-rich element-binding protein, in anti-proliferative effects of metformin in cancer cells. p53 wild-type and p53 mutant breast cancer cells were treated with metformin, and expression of TTP and c-Myc was analyzed by semi-quantitative RT-PCR, Western blots, and promoter activity assay. Breast cancer cells were transfected with siRNA against TTP to inhibit TTP expression or c-Myc and, after metformin treatment, analyzed for cell proliferation by MTS assay. Metformin induces the expression of tristetraprolin (TTP) in breast cancer cells in a p53-independent manner. Importantly, inhibition of TTP abrogated the anti-proliferation effect of metformin. We observed that metformin decreased c-Myc levels, and ectopic expression of c-Myc blocked the effect of metformin on TTP expression and cell proliferation. Our data indicate that metformin induces TTP expression by reducing the expression of c-Myc, suggesting a new model whereby TTP acts as a mediator of metformin's anti-proliferative activity in cancer cells.

Tristetraprolin mediates anti-inflammatory effects of carbon monoxide on lipopolysaccharide-induced acute lung injury.

Low-dose inhaled carbon monoxide is reported to suppress inflammatory responses and exhibit a therapeutic effect in models of lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the precise mechanism by which carbon monoxide confers protection against ALI is not clear. Tristetraprolin (TTP; official name ZFP36) exerts anti-inflammatory effects by enhancing decay of proinflammatory cytokine mRNAs. With the use of TTP knockout mice, we demonstrate here that the protection by carbon monoxide against LPS-induced ALI is mediated by TTP. Inhalation of carbon monoxide substantially increased the pulmonary expression of TTP. carbon monoxide markedly enhanced the decay of mRNA-encoding inflammatory cytokines, blocked the expression of inflammatory cytokines, and decreased tissue damage in LPS-treated lung tissue. Moreover, knockout of TTP abrogated the anti-inflammatory and tissue-protective effects of carbon monoxide in LPS-induced ALI. These results suggest that carbon monoxide-induced TTP mediates the protective effect of carbon monoxide against LPS-induced ALI by enhancing the decay of mRNA encoding proinflammatory cytokines.

Cilostazol attenuates murine hepatic ischemia and reperfusion injury via heme oxygenase-dependent activation of mitochondrial biogenesis.

Hepatic ischemia-reperfusion (I/R) can cause hepatocellular injury associated with the inflammatory response and mitochondrial dysfunction. We studied the protective effects of the phosphodiesterase inhibitor cilostazol in hepatic I/R and the roles of mitochondria and the Nrf2/heme oxygenase-1 (HO-1) system. Wild-type, Hmox1(-/-), or Nrf2(-/-) mice were subjected to hepatic I/R in the absence or presence of cilostazol followed by measurements of liver injury. Primary hepatocytes were subjected to cilostazol with the HO-1 inhibitor ZnPP, or Nrf2-specific siRNA, followed by assessment of mitochondrial biogenesis. Preconditioning with cilostazol prior to hepatic I/R protected against hepatocellular injury and mitochondrial dysfunction. Cilostazol reduced the serum levels of alanine aminotransferase, TNF-α, and liver myeloperoxidase content relative to control I/R-treated mice. In primary hepatocytes, cilostazol increased the expression of HO-1, and markers of mitochondrial biogenesis, PGC-1α, NRF-1, and TFAM, induced the mitochondrial proteins COX III and COX IV and increased mtDNA and mitochondria content. Pretreatment of primary hepatocytes with ZnPP inhibited cilostazol-induced PGC-1α, NRF-1, and TFAM mRNA expression and reduced mtDNA and mitochondria content. Genetic silencing of Nrf2 prevented the induction of HO-1 and mitochondrial biogenesis by cilostazol in HepG2 cells. Cilostazol induced hepatic HO-1 production and mitochondrial biogenesis in wild-type mice, but not in Hmox1(-/-) or Nrf2(-/-) mice, and failed to protect against liver injury in Nrf2(-/-) mice. These results suggest that I/R injury can impair hepatic mitochondrial function, which can be reversed by cilostazol treatment. These results also suggest that cilostazol-induced mitochondrial biogenesis was mediated by an Nrf-2- and HO-1-dependent pathway.

Carbon Monoxide Inhibits Tenascin-C Mediated Inflammation via IL-10 Expression in a Septic Mouse Model.

Tenascin-C (TN-C), an extracellular matrix (ECM) glycoprotein, is specifically induced upon tissue injury and infection and during septic conditions. Carbon monoxide (CO) gas is known to exert various anti-inflammatory effects in various inflammatory diseases. However, the mechanisms underlying the effect of CO on TN-C-mediated inflammation are unknown. In the present study, we found that treatment with LPS significantly enhanced TN-C expression in macrophages. CO gas, or treatment with the CO-donor compound, CORM-2, dramatically reduced LPS-induced expression of TN-C and proinflammatory cytokines while significantly increased the expression of IL-10. Treatment with TN-C siRNA significantly suppressed the effects of LPS on proinflammatory cytokines production. TN-C siRNA did not affect the CORM-2-dependent increase of IL-10 expression. In cells transfected with IL-10 siRNA, CORM-2 had no effect on the LPS-induced expression of TN-C and its downstream cytokines. These data suggest that IL-10 mediates the inhibitory effect of CO on TN-C and the downstream production of proinflammatory cytokines. Additionally, administration of CORM-2 dramatically reduced LPS-induced TN-C and proinflammatory cytokines production while expression of IL-10 was significantly increased. In conclusion, CO regulated IL-10 expression and thus inhibited TN-C-mediated inflammation in vitro and in vivo.

Pretreatment with CO-releasing molecules suppresses hepcidin expression during inflammation and endoplasmic reticulum stress through inhibition of the STAT3 and CREBH pathways.

The circulating peptide hormone hepcidin maintains systemic iron homeostasis. Hepcidin production increases during inflammation and as a result of endoplasmic reticulum (ER) stress. Elevated hepcidin levels decrease dietary iron absorption and promote iron sequestration in reticuloendothelial macrophages. Furthermore, increased plasma hepcidin levels cause hypoferremia and the anemia associated with chronic diseases. The signal transduction pathways that regulate hepcidin during inflammation and ER stress include the IL-6-dependent STAT-3 pathway and the unfolded protein response-associated cyclic AMP response element-binding protein-H (CREBH) pathway, respectively. We show that carbon monoxide (CO) suppresses hepcidin expression elicited by IL-6- and ER-stress agents by inhibiting STAT-3 phosphorylation and CREBH maturation, respectively. The inhibitory effect of CO on IL-6-inducible hepcidin expression is dependent on the suppressor of cytokine signaling-3 (SOCS-3) protein. Induction of ER stress in mice resulted in increased hepatic and serum hepcidin. CO administration inhibited ER-stress-induced hepcidin expression in vivo. Furthermore, ER stress caused iron accumulation in splenic macrophages, which could be prevented by CO. Our findings suggest novel anti-inflammatory therapeutic applications for CO, as well as therapeutic targets for the amelioration of anemia in the hypoferremic condition associated with chronic inflammatory and metabolic diseases.

Induction of heme oxygenase-1 with hemin reduces obesity-induced adipose tissue inflammation via adipose macrophage phenotype switching.

Adipose macrophages with the anti-inflammatory M2 phenotype protect against obesity-induced inflammation and insulin resistance. Heme oxygenase-1 (HO-1), which elicits antioxidant and anti-inflammatory activity, modulates macrophage phenotypes and thus is implicated in various inflammatory diseases. Here, we demonstrate that the HO-1 inducer, hemin, protects against obesity-induced adipose inflammation by inducing macrophages to switch to the M2 phenotype. HO-1 induction by hemin reduced the production of proinflammatory cytokines (TNF-α and IL-6) from cocultured adipocytes and macrophages by inhibiting the activation of inflammatory signaling molecules (JNK and NF-κB) in both cell types. Hemin enhanced transcript levels of M2 macrophage marker genes (IL-4, Mrc1, and Clec10a) in the cocultures, while reducing transcripts of M1 macrophage markers (CD274 and TNF-α). The protective effects of hemin on adipose inflammation and macrophage phenotype switching were confirmed in mice fed a high-fat diet, and these were associated with PPARγ upregulation and STAT6 activation. These findings suggest that induction of HO-1 with hemin protects against obesity-induced adipose inflammation through M2 macrophage phenotype switching, which is induced by the PPARγ and STAT6 pathway. HO-1 inducers such as hemin may be useful for preventing obesity-induced adipose inflammation.