Ja Hyun Koo

AMPK facilitates nuclear accumulation of Nrf2 by phosphorylating at serine 550

ABSTRACT Nrf2 (nuclear factor erythroid 2-related factor 2) is an antioxidant transcription factor. AMP-activated protein kinase (AMPK) functions as a central regulator of cell survival in response to stressful stimuli. Nrf2 should be coordinated with the cell survival pathway controlled by AMPK, but so far the mechanistic connections remain undefined. This study investigated the role of AMPK in Nrf2 trafficking and its activity regulation. A subnetwork integrating neighbor molecules suggested direct interaction between AMPK and Nrf2. In cells, AMPK activation caused nuclear accumulation of Nrf2. In the in vitro kinase and peptide competition assays, AMPK phosphorylated Nrf2 at the Ser558 residue (Ser550 in mouse) located in the canonical nuclear export signal. Nrf2 with an S550A mutation failed to be accumulated in the nucleus after AMPK activation. Leptomycin B, a nuclear export inhibitor, did not enhance nuclear accumulation of wild-type Nrf2 (WT-Nrf2) activated by AMPK or a phospho-Ser550-mimetic Nrf2 mutant, corroborating the finding that AMPK facilitated nuclear accumulation of Nrf2, probably by inhibiting nuclear export. Activated glycogen synthase kinase 3β (GSK3β) diminished the basal nuclear level of Myc-S550A-Nrf2. Taking the data collectively, AMPK phosphorylates Nrf2 at the Ser550 residue, which, in conjunction with AMPK-mediated GSK3β inhibition, promotes nuclear accumulation of Nrf2 for antioxidant response element (ARE)-driven gene transactivation.

Fyn inhibition by cycloalkane-fused 1,2-dithiole-3-thiones enhances antioxidant capacity and protects mitochondria from oxidative injury.

Fyn kinase has emerged as a regulator of diverse pathological processes. However, therapeutic Fyn inhibitors are not available. This study investigated the potential of a series of cycloalkane-fused dithiolethiones (CDTs) or other congeners to increase antioxidant capacity in association with Fyn inhibition, as well as the molecular basis for this effect. Treatment of HepG2 cells with each agent protected the mitochondria from oxidative injury elicited by arachidonic acid and iron, which increased cell viability; 4,5,6,7-tetrahydrobenzo-1,2-dithiole-3-thione (SNU1A) and 5,6-dihydro-4H-cyclopenta-1,2-dithiole-3-thione (SNU2A) were the most effective, whereas 5-methyl-1,2-dithiole-3-thione (SNU3A) was less active. 5-(Quinolin-2-yl)-1,2-dithiole-3-thione (SNU3E) had a minimal effect. SNU1A treatment decreased mitochondrial superoxide production and enabled cells to restore mitochondrial membrane permeability. Oxidative injury caused by arachidonic acid and iron enhanced Fyn phosphorylation at a tyrosine residue, which was decreased by SNU1A treatment. 2,3-Dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide (SU6656), a known Fyn inhibitor, had a similar effect. Fyn inhibition contributed to protecting mitochondria from injury through AMP-activated protein kinase (AMPK), as supported by reversal of this effect with Fyn overexpression. Consistently, Fyn overexpression attenuated AMPK activation by SNU1A, which strengthens the inhibitory role of Fyn in AMPK activity. CDTs had antioxidant effects, as shown by increases in GSH contents and inhibition of H2O2 production. They also had the ability to activate nuclear factor E2–related factor 2 (Nrf2), a key antioxidant transcription factor. Fyn overexpression decreased the Nrf2 activation induced by SNU1A. Our results demonstrate that CDTs exert cytoprotective effects by protecting mitochondria and increasing the cellular antioxidant capacity, which may result not only from Fyn inhibition leading to AMPK activation but also from Nrf2 activation.

PHLDA3 overexpression in hepatocytes by endoplasmic reticulum stress via IRE1–Xbp1s pathway expedites liver injury

Objective Endoplasmic reticulum (ER) stress is involved in liver injury, but molecular determinants are largely unknown. This study investigated the role of pleckstrin homology-like domain, family A, member-3 (PHLDA3), in hepatocyte death caused by ER stress and the regulatory basis. Design Hepatic PHLDA3 expression was assessed in HCV patients with hepatitis and in several animal models with ER stress. Immunoblottings, PCR, reporter gene, chromatin immunoprecipitation (ChIP) and mutation analyses were done to explore gene regulation. The functional effect of PHLDA3 on liver injury was validated using lentiviral delivery of shRNA. Results PHLDA3 was overexpressed in relation to hepatocyte injury in patients with acute liver failure or liver cirrhosis or in toxicant-treated mice. In HCV patients with liver injury, PHLDA3 was upregulated in parallel with the induction of ER stress marker. Treatment of mice with tunicamycin (Tm) (an ER stress inducer) increased PHLDA3 expression in the liver. X box-binding protein-1 (Xbp1) was newly identified as a transcription factor responsible for PHLDA3 expression. Inositol-requiring enzyme 1 (IRE1) (an upstream regulator of Xbp1) was required for PHLDA3 induction by Tm, whereas other pathways (c-Jun N-terminal kinase (JNK), protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6)) were not. PHLDA3 overexpression correlated with the severity of hepatocyte injury in animal or cell model of ER stress. In p53-deficient cells, ER stress inducers transactivated PHLDA3 with a decrease in cell viability. ER stress-induced hepatocyte death depended on serine/threonine protein kinase B (Akt) inhibition by PHLDA3. Lentiviral delivery of PHLDA3 shRNA to mice abrogated p-Akt inhibition in the liver by Tm, attenuating hepatocyte injury. Conclusions ER stress in hepatocytes induces PHLDA3 via IRE1–Xbp1s pathway, which facilitates liver injury by inhibiting Akt.

Hepcidin inhibits Smad3 phosphorylation in hepatic stellate cells by impeding ferroportin-mediated regulation of Akt

Hepatic stellate cell (HSC) activation on liver injury facilitates fibrosis. Hepatokines affecting HSCs are largely unknown. Here we show that hepcidin inhibits HSC activation and ameliorates liver fibrosis. We observe that hepcidin levels are inversely correlated with exacerbation of fibrosis in patients, and also confirm the relationship in animal models. Adenoviral delivery of hepcidin to mice attenuates liver fibrosis induced by CCl4 treatment or bile duct ligation. In cell-based assays, either hepcidin from hepatocytes or exogenous hepcidin suppresses HSC activation by inhibiting TGFβ1-mediated Smad3 phosphorylation via Akt. In activated HSCs, ferroportin is upregulated, which can be prevented by hepcidin treatment. Similarly, ferroportin knockdown in HSCs prohibits TGFβ1-inducible Smad3 phosphorylation and increases Akt phosphorylation, whereas ferroportin over-expression has the opposite effect. HSC-specific ferroportin deletion also ameliorates liver fibrosis. In summary, hepcidin suppresses liver fibrosis by impeding TGFβ1-induced Smad3 phosphorylation in HSCs, which depends on Akt activated by a deficiency of ferroportin.

Farnesoid X receptor as a regulator of fuel consumption and mitochondrial function

Maintenance of energy homeostasis is crucial for survival of organism. There exists a close link between energy metabolism and cell survival, which are coordinately regulated by common signaling pathways. Farnesoid X receptor (FXR) serves as a ligand-mediated transcription factor to regulate diverse genes involved in bile acid, lipid, and glucose metabolism, controlling cellular and systemic energy metabolism. Another important aspect on FXR biology is related to its beneficial effect on cell survival. FXR exerts antioxidative and cytoprotective effect, which is closely associated with the ability of FXR to regulate mitochondrial function. To maintain complex biological processes under homeostasis, FXR activity needs to be dynamically and tightly controlled by different signaling pathways and modifications. In this review, we discuss the role of FXR in the regulation of energy metabolism and cell survival, with the goal of understanding molecular basis for FXR regulation in physiological and pathological conditions. This information may be of assistance in understanding recent advancements of FXR research and strategies for the prevention and treatment of metabolic disorders.

Phytochemical regulation of Fyn and AMPK signaling circuitry

During the past decades, phytochemical terpenoids, polyphenols, lignans, flavonoids, and alkaloids have been identified as antioxidative and cytoprotective agents. Adenosine monophosphate-activated protein kinase (AMPK) is a kinase that controls redox-state and oxidative stress in the cell, and serves as a key molecule regulating energy metabolism. Many phytochemicals directly or indirectly alter the AMPK pathway in distinct manners, exerting catabolic metabolism. Some of them are considered promising in the treatment of metabolic diseases such as type II diabetes, obesity, and hyperlipidemia. Another important kinase that regulates energy metabolism is Fyn kinase, a member of the Src family kinases that plays a role in various cellular responses such as insulin signaling, cell growth, oxidative stress and apoptosis. Phytochemical inhibition of Fyn leads to AMPK-mediated protection of the cell in association with increased antioxidative capacity and mitochondrial biogenesis. The kinases may work together to form a signaling circuitry for the homeostasis of energy conservation and expenditure, and may serve as targets of phytochemicals. This review is intended as a compilation of recent advancements in the pharmacological research of phytochemicals targeting Fyn and AMPK circuitry, providing information for the prevention and treatment of metabolic diseases and the accompanying tissue injuries.

Gα12 overexpression induced by miR-16 dysregulation contributes to liver fibrosis by promoting autophagy in hepatic stellate cells

BACKGROUND & AIMS Hepatic stellate cells (HSCs) have a role in liver fibrosis. Guanine nucleotide-binding α-subunit 12 (Gα12) converges signals from G-protein-coupled receptors whose ligand levels are elevated in the environment during liver fibrosis; however, information is lacking on the effect of Gα12 on HSC trans-differentiation. This study investigated the expression of Gα12 in HSCs and the molecular basis of the effects of its expression on liver fibrosis. METHODS Gα12 expression was assessed by immunostaining, and immunoblot analyses of mouse fibrotic liver tissues and primary HSCs. The role of Gα12 in liver fibrosis was estimated using a toxicant injury mouse model with Gα12 gene knockout and/or HSC-specific Gα12 delivery using lentiviral vectors, in addition to primary HSCs and LX-2 cells using microRNA (miR) inhibitors, overexpression vectors, or adenoviruses. miR-16, Gα12, and LC3 were also examined in samples from patients with fibrosis. RESULTS Gα12 was overexpressed in activated HSCs and fibrotic liver, and was colocalised with desmin. In a carbon tetrachloride-induced fibrosis mouse model, Gα12 ablation prevented increases in fibrosis and liver injury. This effect was attenuated by HSC-specific lentiviral delivery of Gα12. Moreover, Gα12 activation promoted autophagy accompanying c-Jun N-terminal kinase-dependent ATG12-5 conjugation. In addition, miR-16 was found to be a direct inhibitor of the de novo synthesis of Gα12. Modulations of miR-16 altered autophagy in HSCs. In a fibrosis animal model or patients with severe fibrosis, miR-16 levels were lower than in their corresponding controls. Consistently, cirrhotic patient liver tissues showed Gα12 and LC3 upregulation in desmin-positive areas. CONCLUSIONS miR-16 dysregulation in HSCs results in Gα12 overexpression, which activates HSCs by facilitating autophagy through ATG12-5 formation. This suggests that Gα12 and its regulatory molecules could serve as targets for the amelioration of liver fibrosis. LAY SUMMARY Guanine nucleotide-binding α-subunit 12 (Gα12) is upregulated in activated hepatic stellate cells (HSCs) as a consequence of the dysregulation of a specific microRNA that is abundant in HSCs, facilitating the progression of liver fibrosis. This event is mediated by c-Jun N-terminal kinase-dependent ATG12-5 formation and the promotion of autophagy. We suggest that Gα12 and its associated regulators could serve as new targets in HSCs for the treatment of liver fibrosis.

Gα13 ablation reprograms myofibers to oxidative phenotype and enhances whole-body metabolism

Skeletal muscle is a key organ in energy homeostasis owing to its high requirement for nutrients. Heterotrimeric G proteins converge signals from cell-surface receptors to potentiate or blunt responses against environmental changes. Here, we show that muscle-specific ablation of Gα13 in mice promotes reprogramming of myofibers to the oxidative type, with resultant increases in mitochondrial biogenesis and cellular respiration. Mechanistically, Gα13 and its downstream effector RhoA suppressed nuclear factor of activated T cells 1 (NFATc1), a chief regulator of myofiber conversion, by increasing Rho-associated kinase 2-mediated (Rock2-mediated) phosphorylation at Ser243. Ser243 phosphorylation of NFATc1 was reduced after exercise, but was higher in obese animals. Consequently, Gα13 ablation in muscles enhanced whole-body energy metabolism and increased insulin sensitivity, thus affording protection from diet-induced obesity and hepatic steatosis. Our results define Gα13 as a switch regulator of myofiber reprogramming, implying that modulations of Gα13 and its downstream effectors in skeletal muscle are a potential therapeutic approach to treating metabolic diseases.

Gα12 ablation exacerbates liver steatosis and obesity by suppressing USP22/SIRT1-regulated mitochondrial respiration

Nonalcoholic fatty liver disease (NAFLD) arises from mitochondrial dysfunction under sustained imbalance between energy intake and expenditure, but the underlying mechanisms controlling mitochondrial respiration have not been entirely understood. Heterotrimeric G proteins converge with activated GPCRs to modulate cell-signaling pathways to maintain metabolic homeostasis. Here, we investigated the regulatory role of G protein &agr;12 (G&agr;12) on hepatic lipid metabolism and whole-body energy expenditure in mice. Fasting increased G&agr;12 levels in mouse liver. G&agr;12 ablation markedly augmented fasting-induced hepatic fat accumulation. cDNA microarray analysis from Gna12-KO liver revealed that the G&agr;12-signaling pathway regulated sirtuin 1 (SIRT1) and PPAR&agr;, which are responsible for mitochondrial respiration. Defective induction of SIRT1 upon fasting was observed in the liver of Gna12-KO mice, which was reversed by lentivirus-mediated G&agr;12 overexpression in hepatocytes. Mechanistically, G&agr;12 stabilized SIRT1 protein through transcriptional induction of ubiquitin-specific peptidase 22 (USP22) via HIF-1&agr; increase. G&agr;12 levels were markedly diminished in liver biopsies from NAFLD patients. Consistently, Gna12-KO mice fed a high-fat diet displayed greater susceptibility to diet-induced liver steatosis and obesity due to decrease in energy expenditure. Our results demonstrate that G&agr;12 regulates SIRT1-dependent mitochondrial respiration through HIF-1&agr;–dependent USP22 induction, identifying G&agr;12 as an upstream molecule that contributes to the regulation of mitochondrial energy expenditure.

Gα12 regulates osteoclastogenesis by modulating NFATc1 expression

The G12 family of G protein alpha subunits has been shown to participate in the regulation of various physiological processes. However, the role of Gα12 in bone physiology has not been well described. Here, by micro‐CT analysis, we discovered that Gα12‐knockout mice have an osteopetrotic phenotype. Histological examination showed lower osteoclast number in femoral tissue of Gα12‐knockout mice compared to wild‐type mice. Additionally, in vitro osteoclastic differentiation of precursor cells with receptor activator of nuclear factor‐κB ligand (RANKL) showed that Gα12 deficiency decreased the number of osteoclast generated and the bone resorption activity. The induction of nuclear factor of activated T‐cell c1 (NFATc1), the key transcription factor of osteoclastogenesis, and the activation of RhoA by RANKL was also significantly suppressed by Gα12 deficiency. We further found that the RANKL induction of NFATc1 was not dependent on RhoA signalling, while osteoclast precursor migration and bone resorption required RhoA in the Gα12‐mediated regulation of osteoclasts. Therefore, Gα12 plays a role in differentiation through NFATc1 and in cell migration and resorption activity through RhoA during osteoclastogenesis.