Sang Ick Park

AMP-activated protein kinase inhibits TGF-β-induced fibrogenic responses of hepatic stellate cells by targeting transcriptional coactivator p300

Liver fibrosis is a common consequence of various chronic liver injuries, including virus infection and ethanol. Activated hepatic stellate cells (HSCs) contribute to liver fibrosis through the accumulation of extracellular matrix proteins, including type I alpha collagen (COL1A). The activation of adenosine monophosphate‐activated protein kinase (AMPK) modulates HSCs activation, but its underlying mechanism remains unclear. Here, we report that AMPK inhibits transforming growth factor (TGF)‐β‐induced fibrogenic property of HSCs by regulating transcriptional coactivator p300. We treated human (LX‐2) and rat (CFSC‐2G) HSC lines with TGF‐β to induce fibrogenic activation of HSCs. Pharmacological activation of AMPK by treatment with 5‐aminoimidazole‐4‐carboxamide‐1‐beta‐4‐ribofuranoside (AICAR), metformin, or adiponectin lowered TGF‐β‐induced expression of COL1A and myofibroblast marker alpha‐smooth muscle actin (α‐SMA). Transient transduction of constitutively active AMPKα (caAMPKα) was sufficient to attenuate COL1A and α‐SMA expression, whereas an AMPK inhibitor considerably abrogated the inhibitory effect of AICAR on fibrogenic gene expression. Although AMPK significantly suppressed Smad‐dependent transcription, it did not affect TGF‐β‐stimulated phosphorylation, nuclear localization, or DNA‐binding activity of Smad2/3. AICAR rather attenuated TGF‐β‐induced Smad3 interaction with transcriptional coactivator p300 accompanying with reduction of Smad3 acetylation. Moreover, AICAR induced not only physical interaction between AMPK and p300 but also proteasomal degradation of p300 protein. Our data provide substantial evidence that AMPK could be a novel therapeutic target for treatment of liver fibrosis, by demonstrating the underlying mechanism of AMPK‐induced antifibrotic function in HSCs. J. Cell. Physiol. 227: 1081–1089, 2012.

HBx-Induced Hepatic Steatosis and Apoptosis Are Regulated by TNFR1- and NF-κB-Dependent Pathways

Hepatitis B virus X (HBx) protein is an important regulator of hepatic steatosis observed in patients with hepatitis B virus; however, its underlying molecular mechanism remains unclear. TNF receptor 1 (TNFR1) is an essential pathway for the HBx-mediated nuclear factor kappaB (NF-kappaB) activation involved in hepatic liver injury. Here, we show that HBx-mediated steatosis and apoptosis are regulated by TNFR1- and NF-kappaB-dependent pathways. HBx-mediated tumor necrosis factor alpha (TNF-alpha) production and NF-kappaB activation were completely diminished in anti-TNF-alpha-treated cells and TNF-alpha(-)(/-) or TNFR1(-/-) mice. HBx and TNFR1, which are potentiated by TNF-alpha, are physically associated and colocalize in the plasma membrane. Similarly, TNFR1 depletion inhibits lipid droplets, and lipogenic genes such as sterol regulatory element binding protein (SREBP) 1 and peroxisome proliferator-activated receptor (PPAR) gamma increased in HBx-Tg mice and HepG2-GFPHBx stable cells. Furthermore, lipid accumulation and expression of SREBP1c and PPAR gamma are significantly increased in AdHBx-GFP-injected (intravenous) wild-type mice, but not in TNFR1(-/-) mice. HBx-enhanced transcriptional activities of SREBP1 and PPAR gamma are significantly attenuated by the NF-kappaB inhibitor Bay 11-7082, as well as by TNFR1 depletion. Also, AdHBx-GFP potentiates TNF-alpha-induced apoptosis, which is completely inhibited in TNFR1-depleted cells. Our results suggest that HBx-induced NF-kappaB activation was mediated by direct interaction with TNFR1 and thereby induced TNF-alpha production. HBx-induced NF-kappaB activation is also associated with the induction of hepatic steatosis and apoptosis, which is determined by a TNFR1-dependent pathway.

A critical role of STAT1 in streptozotocin-induced diabetic liver injury in mice: controlled by ATF3.

It is well-established that the administration of streptozotocin accelerates diabetic liver injury as well as type-I diabetes, however the underlying mechanisms are poorly understood. Here we investigated the molecular mechanisms of diabetic liver injury in a model of streptozotocin (STZ)-induced type-I diabetes. STZ administration induced type-1 diabetes and chronic liver injury was associated with increased STAT1, which is implicated in diabetic liver injury by virtue of its ability to promote hepatocyte apoptosis, in the liver and pancreas, which were all strongly inhibited in STAT1(-)(/-) mice. Similarly, STZ-induced ATF3, a stress-inducible gene, was completely abolished in the liver of IFN-gamma(-/-) mice, but not in STAT1(-/-) mice. Inhibition of STAT1 by siRNA or dominant-negative DNA did not affect ATF3 protein expression but blocked IFN-gamma-induced ATF3 translocation from the cytosol into the nucleus. In contrast, inhibition of ATF3 by using siRNA diminished STAT1 protein expression and IFN-gamma/STZ-induced hepatocyte apoptosis. Furthermore, GST pull-down and co-IP assay showed that STAT1 bound to C-terminal domain of ATF3. Such direct interaction increased the stability of STAT1 by inhibiting its ubiquitination as well as proteasome activity. Our results suggest that STAT1 is a common signaling pathway contributing to STZ-induced diabetes and diabetic liver injury. ATF3 functions as a potent regulator of STAT1 stability, accelerating STZ-induced diabetes and diabetic liver injury.

GO6976 prevents TNF-α-induced suppression of adiponectin expression in 3T3-L1 adipocytes: Putative involvement of protein kinase C

Adiponectin, one of the adipokines secreted by adipocytes, possesses insulin sensitizing and anti‐atherosclerotic properties. Tumor necrosis factor‐alpha (TNF‐α) is known to suppress the expression and secretion of adiponectin in adipocytes; however, the underlying mechanism remains poorly understood. Here, we demonstrate that GO6976 (a selective inhibitor of conventional protein kinase C (PKC)) prevents TNF‐α‐induced suppression of adiponectin secretion and expression in fully differentiated 3T3‐L1 adipocytes, accompanied by attenuation of c‐Jun N‐terminal kinase (JNK) activation. Additionally, the transcriptional activity of peroxisome proliferator‐activated receptor‐gamma (PPARg) (a strong inducer of adiponectin) on the adiponectin promoter was inhibited in a PKC isoform‐specific manner. These results raise the possibility that PKC is involved in TNF‐α‐induced suppression of adiponectin in 3T3‐L1 adipocytes.

Induction of Id2 expression by cardiac transcription factors GATA4 and Nkx2.5

Inhibitor of differentiation/DNA binding (Id) proteins function as a regulator of helix‐loop‐helix proteins participating in cell lineage commitment and differentiation. Here, we observed a marked induction of Id2 during cardiomyocyte differentiation from P19CL6 murine embryonic teratocarcinoma stem cells, prompting us to investigate the upstream regulatory mechanism of Id2 induction. Computer analysis of Id2 promoter and subsequent electrophoretic mobility shift assay revealed several binding sites for GATA4 and Nkx2.5 within the Id2 promoter. By further deletion and mutation analysis of the respective binding site, we identified that two motifs located at −497/−502 and −264/−270 were functionally important for Id2 promoter activation by GATA4 and Nkx2.5, respectively. Overexpression of GATA4 and/or Nkx2.5 induced not only Id2 promoter activity but also Id2 protein expression. Additionally, Id proteins significantly inhibit the GATA4 and Nkx2.5‐dependent transcription, suggesting Id proteins may play a regulatory role in cardiogenesis. Collectively, our results demonstrate that GATA4 and Nkx2.5 could be one of the upstream regulators of Id2. J. Cell. Biochem. 103: 182–194, 2008.

Role of Activating Transcription Factor 3 on TAp73 Stability and Apoptosis in Paclitaxel-Treated Cervical Cancer Cells

Taxol (paclitaxel) is a potent anticancer drug that has been found to be effective against several tumor types, including cervical cancer. However, the exact mechanism underlying the antitumor effects of paclitaxel is poorly understood. Here, paclitaxel induced the apoptosis of cervical cancer HeLa cells and correlated with the enhanced activation of caspase-3 and TAp73, which was strongly inhibited by TAp73β small interfering RNA (siRNA). In wild-type activating transcription factor 3 (ATF3)–overexpressed cells, paclitaxel enhanced apoptosis through increased α and β isoform expression of TAp73; however, these events were attenuated in cells containing inactive COOH-terminal–deleted ATF3 [ATF3(ΔC)] or ATF3 siRNA. In contrast, paclitaxel-induced ATF3 expression did not change in TAp73β-overexpressed or TAp73β siRNA–cotransfected cells. Furthermore, paclitaxel-induced ATF3 translocated into the nucleus where TAp73β is expressed, but not in ATF3(ΔC) or TAp73β siRNA–transfected cells. As confirmed by the GST pull-down assay, ATF3 bound to the DNA-binding domain of p73, resulting in the activation of p21 or Bax transcription, a downstream target of p73. Overexpression of ATF3 prolonged the half-life of TAp73β by inhibiting its ubiquitination and thereby enhancing its transactivation and proapoptotic activities. Additionally, ATF3 induced by paclitaxel potentiated the stability of TAp73β, not its transcriptional level. Chromatin immunoprecipitation analyses show that TAp73β and ATF3 are recruited directly to the p21 and Bax promoter. Collectively, these results reveal that overexpression of ATF3 potentiates paclitaxel-induced apoptosis of HeLa cells, at least in part, by enhancing TAp73βs stability and its transcriptional activity. The investigation shows that ATF3 may function as a tumor-inhibiting factor through direct regulatory effects on TAp73β, suggesting a functional link between ATF3 and TAp73β. (Mol Cancer Res 2008;6(7):1232–49)

Effect of excess iron on oxidative stress and gluconeogenesis through hepcidin during mitochondrial dysfunction

Excessive tissue iron levels are a risk factor for insulin resistance and type 2 diabetes, which are associated with alterations in iron metabolism. However, the mechanisms underlying this association are not well understood. This study used human liver SK-HEP-1 cells to examine how excess iron induces mitochondrial dysfunction and how hepcidin controls gluconeogenesis. Excess levels of reactive oxygen species (ROS) and accumulated iron due to iron overload induced mitochondrial dysfunction, leading to a decrease in cellular adenosine triphosphate content and cytochrome c oxidase III expression, with an associated increase in gluconeogenesis. Disturbances in mitochondrial function caused excess iron deposition and unbalanced expression of iron metabolism-related proteins such as hepcidin, ferritin H and ferroportin during the activation of p38 mitogen-activated protein kinase (MAPK) and CCAAT/enhancer-binding protein alpha (C/EBPα), which are responsible for increased phosphoenolpyruvate carboxykinase expression. Desferoxamine and n-acetylcysteine ameliorated these deteriorations by inhibiting p38 MAPK and C/EBPα activity through iron chelation and ROS scavenging activity. Based on experiments using hepcidin shRNA and hepcidin overexpression, the activation of hepcidin affects ROS generation and iron deposition, which disturbs mitochondrial function and causes an imbalance in iron metabolism and increased gluconeogenesis. Repression of hepcidin activity can reverse these changes. Our results demonstrate that iron overload is associated with mitochondrial dysfunction and that together they can cause abnormal hepatic gluconeogenesis. Hepcidin expression may modulate this disorder by regulating ROS generation and iron deposition.

Association of circulating irisin levels with metabolic and metabolite profiles of Korean adolescents

CONTEXT Irisin, a novel exercise-induced myokine, has been suggested to regulate energy metabolism. OBJECTIVE We studied the relationship between circulating irisin and metabolic and metabolite profiles of Korean adolescents, and investigated the effects of physical activity, obesity, and metabolic syndrome (MetS) on irisin levels. MATERIALS AND METHODS Data were obtained from the Korean Children-Adolescents Study. Our cross-sectional study included 618 adolescents (370 normal-weight and 248 obese adolescents; 316 boys and 302 girls) aged 12-15years. Body composition was determined using an impedance body composition analyzer and general participant characteristics and lifestyle information were obtained from questionnaires. Serum irisin levels were measured using a commercial kit. RESULTS Mean body mass index (BMI) was 19.4kg/m2 in normal-weight adolescents and 31.4kg/m2 in obese adolescents. Circulating irisin was positively correlated with adiposity indices, including BMI z-score, waist circumference, percent body fat, fat mass, fat-free mass, fat mass to fat-free mass ratio, and lipid and glucose metabolism markers, including total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglycerides, glucose, insulin, and homeostasis model assessment-estimated insulin resistance (all p≤0.006). Of these, increased body fat mass [standardized (Std) ß, 0.23; p<0.0001], LDL-C (Std ß, 0.14; p=0.0005) and fasting glucose (Std ß, 0.08; p=0.0383) were the main independent factors associated with higher irisin levels. Moreover, elevated serum irisin was associated with the risk of obesity [odds ratio (OR], 2.2; confidence interval (CI), 1.19-3.87] and MetS (OR, 2.0; CI, 1.15-3.47). Furthermore, irisin and branched-chain amino acids were positively associated (p<4×10-4 for Bonferroni correction). Additionally, in the normal-weight group, girls had higher irisin levels than boys (p=0.006) and adolescents who engaged in regular physical activity had higher levels of irisin than sedentary adolescents (p=0.0388). The relationship between physical activity and irisin levels was not observed in obese adolescents. CONCLUSIONS Elevated serum irisin was independently associated with the risk of obesity and positively correlated with unhealthy metabolic parameters and metabolites. Moreover, irisin levels were higher in active versus sedentary adolescents in the normal-weight group, but not in the obese group. Our findings suggest that irisin plays an important role in metabolic disorders and may be affected by physiopathological status.

Nitrated fatty acids prevent TNFα-stimulated inflammatory and atherogenic responses in endothelial cells

Nitration products (nitroalkenes) of linoleic acid (LNO(2)) and oleic acid (OA-NO(2)) can act as endogenous PPARgamma ligands with electrophilic properties to exert anti-inflammatory effects on atherosclerotic plaques in the vasculature. Here, we show that OA-NO(2) and LNO(2) prevent tumor necrosis factor alpha (TNFalpha)-stimulated inflammatory and atherogenic responses in human umbilical vein endothelial cells (HUVECs). Both OA-NO(2) and LNO(2) prevented TNFalpha-stimulated release of the cytokines, IL-6, IL-8, IL-12/p40, IFNgamma, MCP-1, and IP-10, and inhibited NF-kappaB activation. OA-NO(2) and LNO(2) also blocked TNFalpha-induced expression of the adhesion molecules, ICAM-1, VCAM-1, and E-selectin, and suppressed monocyte adhesion to HUVECs. In each case, OA-NO(2) was more potent and efficacious than was LNO(2), possibly due to increased stability in aqueous media. Collectively, these results substantiate a new functional role for nitrated fatty acids, demonstrating that OA-NO(2) and LNO(2) exert an anti-inflammatory function against the inflammatory cascade initiated by the representative pro-inflammatory cytokine, TNFalpha.

TRAIL regulates collagen production through HSF1-dependent Hsp47 expression in activated hepatic stellate cells

Hsp47 is a collagen-specific molecular chaperone, whose activity has been implicated in liver fibrosis. In this study, we showed that TRAIL treatment inhibited Hsp47 expression in dose- and time-dependent manners, subsequently leading to the decrease of collagen production in activated human hepatic stellate LX-2 cells. Overexpression of Hsp47 in LX-2 cells acquired resistance for TRAIL-induced collagen reduction and conversely, siRNA suppression of Hsp47 enhanced the decrease of collagen production due to TRAIL treatment. Moreover, we found that Hsp47 expression was under the transcriptional control of heat shock factor (HSF) 1 which is highly located on nucleus in activated human hepatic stellate LX-2 cells. Treatment of LX-2 cells with TRAIL decreased the active trimer formation of HSF1, increased the dephosphorylation of HSF1 (Ser(230)), and enhanced the translocation of HSF1 into cytosol. The accumulated HSF1 in cytosol led to downregulation of Hsp47 expression, resulting in the reduction of collagen production. Consistently, HSF1 silencing by siRNA prevented Hsp47 induction and subsequent collagen production, whereas overexpression of HSF1 restored the expression level of Hsp47 as well as collagen production in response to TRAIL treatment in LX-2 cells. Taken together, our data suggested that TRAIL induced HSF1 inactivation, consequently leading to the suppression of Hsp47-dependent collagen production in activated human hepatic stellate cells. Therefore, this study suggests that TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis.