A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron.

Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8+ T cell response, and triggers macrophage 'nutritional immunity' to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into 'oncobiotics' for a multi-layered approach to cancer therapy.

Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum.

Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction.

Critical regulation of follicular helper T cell differentiation and function by Gα13 signaling.

GPCR-Gα protein-mediated signal transduction contributes to spatiotemporal interactions between immune cells to fine-tune and facilitate the process of inflammation and host protection. Beyond this, however, how Gα proteins contribute to the helper T cell subset differentiation and adaptive response have been underappreciated. Here, we found that Gα13 signaling in T cells plays a crucial role in inducing follicular helper T (Tfh) cell differentiation in vivo. T cell-specific Gα13-deficient mice have diminished Tfh cell responses in a cell-intrinsic manner in response to immunization, lymphocytic choriomeningitis virus infection, and allergen challenges. Moreover, Gα13-deficient Tfh cells express reduced levels of Bcl-6 and CXCR5 and are functionally impaired in their ability to adhere to and stimulate B cells. Mechanistically, Gα13-deficient Tfh cells harbor defective Rho-ROCK2 activation, and Rho agonist treatment recuperates Tfh cell differentiation and expression of Bcl-6 and CXCR5 in Tfh cells of T cell-specific Gα13-deficient mice. Conversely, ROCK inhibitor treatment hampers Tfh cell differentiation in wild-type mice. These findings unveil a crucial regulatory role of Gα13-Rho-ROCK axis in optimal Tfh cell differentiation and function, which might be a promising target for pharmacologic intervention in vaccine development as well as antibody-mediated immune disorders.

Intestinal Inflammation and Regeneration-Interdigitating Processes Controlled by Dietary Lipids in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a disease of chronic inflammatory conditions of the intestinal tract due to disturbance of the inflammation and immune system. Symptoms of IBD include abdominal pain, diarrhea, bleeding, reduced weight, and fatigue. In IBD, the immune system attacks the intestinal tract's inner wall, causing chronic inflammation and tissue damage. In particular, interlukin-6 and interlukin-17 act on immune cells, including T cells and macrophages, to amplify the immune responses so that tissue damage and morphological changes occur. Of note, excessive calorie intake and obesity also affect the immune system due to inflammation caused by lipotoxicity and changes in lipids supply. Similarly, individuals with IBD have alterations in liver function after sustained high-fat diet feeding. In addition, excess dietary fat intake, along with alterations in primary and secondary bile acids in the colon, can affect the onset and progression of IBD because inflammatory cytokines contribute to insulin resistance; the factors include the release of inflammatory cytokines, oxidative stress, and changes in intestinal microflora, which may also contribute to disease progression. However, interfering with de novo fatty acid synthase by deleting the enzyme acetyl-CoA-carboxylase 1 in intestinal epithelial cells (IEC) leads to the deficiency of epithelial crypt structures and tissue regeneration, which seems to be due to Lgr5+ intestinal stem cell function. Thus, conflicting reports exist regarding high-fat diet effects on IBD animal models. This review will focus on the pathological basis of the link between dietary lipids intake and IBD and will cover the currently available pharmacological approaches.

NRF2-mediated SIRT3 induction protects hepatocytes from ER stress-induced liver injury.

Chronic endoplasmic reticulum (ER) stress in hepatocytes plays a role in the pathogenesis of nonalcoholic fatty liver disease. Therefore, given the association between oxidative stress, mitochondrial dysfunction, and ER stress, our study investigated the role of NRF2-mediated SIRT3 activation in ER stress. SIRT3, a sirtuin, was predicted as the target of NRF2 based on bioinformatic analyses and animal experiments. Nrf2 abrogation diminished mitochondrial DNA content in hepatocytes with Ppargc1α and Cpt1a inhibition, whereas its overexpression enhanced oxygen consumption. Further, chromatin immunoprecipitation and luciferase reporter assays indicated that NRF2 induced SIRT3 through the antioxidant responsive element (ARE) sites comprising the -641 to -631 bp and -419 to -409 bp regions. In tunicamycin-induced ER stress conditions and liver injury animal models following ER stress, NRF2 levels were highly correlated with SIRT3. Nrf2 deficiency enhanced the tunicamycin-mediated induction of CHOP, which was attenuated by Sirt3 overexpression. Further, Sirt3 delivery to hepatocytes in Nrf2 knockout mice prevented tunicamycin from increasing mortality by decreasing ER stress. SIRT3 was upregulated in livers of patients with nonalcoholic liver diseases, whereas lower SIRT3 expression coincided with more severe disease conditions. Taken together, our findings indicated that NRF2-mediated SIRT3 induction protects hepatocytes from ER stress-induced injury, which may contribute to the inhibition of liver disease progression.

Plk2-mediated phosphorylation and translocalization of Nrf2 activates anti-inflammation through p53/Plk2/p21cip1 signaling in acute kidney injury.

The Plk2 is a cellular stress-responsive factor that is induced in response to oxidative stress. However, the roles of Plk2 in acute kidney injury (AKI) have not been clarified. We previously found that Plk2 is an interacting factor of Nrf2 in response to cellular stress, since Plk2 is upregulated in the Nrf2-dependent network. Here, we show that the levels of p53, Plk2, p21cip1, and chromatin-bound Nrf2 were all upregulated in kidney tissues of mice or NRK52E cells treated with either cisplatin or methotrexate. Upregulation of Plk2 by p53 led to an increase of Nrf2 in both soluble and chromatin fractions in cisplatin-treated NRK52E cells. Consistently, depletion of Plk2 suppressed the levels of Nrf2. Of note, Plk2 directly phosphorylated Nrf2 at Ser40, which facilitated its interaction with p21cip1 and translocation into the nuclei for the activation of anti-oxidative and anti-inflammatory factors in response to AKI. Together, these findings suggest that Plk2 may serve as an anti-oxidative and anti-inflammatory regulator through the phosphorylation and activation of Nrf2 to protect kidney cells from kidney toxicants and that Plk2 and Nrf2 therefore work cooperatively for the protection and survival of kidney cells from harmful stresses.

Knockout of Purinergic P2Y6 Receptor Fails to Improve Liver Injury and Inflammation in Non-Alcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y6 receptor (P2Y6R) is a pro-inflammatory Gq/G12 family protein-coupled receptor and reportedly contributes to intestinal inflammation and cardiovascular fibrosis, but its role in liver pathogenesis is unknown. Human genomics data analysis revealed that the liver P2Y6R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y6R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y6R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y6R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y6R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y6R may not contribute to the progression of liver injury, despite increased expression in NASH liver.

Dysregulated Liver Metabolism and Polycystic Ovarian Syndrome.

A significant fraction of couples around the world suffer from polycystic ovarian syndrome (PCOS), a disease defined by the characteristics of enhanced androgen synthesis in ovarian theca cells, hyperandrogenemia, and ovarian dysfunction in women. Most of the clinically observable symptoms and altered blood biomarker levels in the patients indicate metabolic dysregulation and adaptive changes as the key underlying mechanisms. Since the liver is the metabolic hub of the body and is involved in steroid-hormonal detoxification, pathological changes in the liver may contribute to female endocrine disruption, potentially through the liver-to-ovary axis. Of particular interest are hyperglycemic challenges and the consequent changes in liver-secretory protein(s) and insulin sensitivity affecting the maturation of ovarian follicles, potentially leading to female infertility. The purpose of this review is to provide insight into emerging metabolic mechanisms underlying PCOS as the primary culprit, which promote its incidence and aggravation. Additionally, this review aims to summarize medications and new potential therapeutic approaches for the disease.

ERα inhibits mesenchymal and amoeboidal movement of liver cancer cell via Gα12.

Hepatocellular carcinoma (HCC) is the second most common cancer worldwide, demonstrating aggressiveness and mortality more frequently in men than in women. Despite reports regarding the inhibitory ability of estrogen receptor alpha (ERα, ESR1) in certain cancer progression, targets and the basis of underlying gender disparity in HCC worsening remain elusive. Here, we report the ability of ERα to transcriptionally inhibit G protein subunit alpha 12 (Gα12) responsible for HCC worsening. First, using human samples and public database, the expression of ERα and Gα12 in HCC was examined. Then, quantitative real-time PCR, chromatin immunoprecipitation-assay, luciferase assay and immunoblottings of liver cancer cell lines confirmed the inhibitory ability of ERα on Gα12 and HCC progression. Gα12 promoted mesenchymal characteristics and amoeboidal movement, which was antagonized by ERα overexpression. Additionally, we found microRNA-141 and microRNA-200a as downstream targets of the Gα12 signaling axis for cancer malignancy regulation under the control of ERα. As for in-depth mechanism, PTP4A1 was found to be directly inhibited by microRNA-141 and microRNA-200a. Moreover, we found the inhibitory effect of ERα on amoeboidal movement by analyzing the morphology and blebbing of liver cancer cells and the active form of MLC levels. The identified targets and ESR1 levels are inversely correlated with human specimens, as well as with sex-biased survival rates of HCC patients. Collectively, ERα-dependent repression of Gα12 and consequent changes in the Gα12 signaling may explain the gender disparity in HCC, providing pharmacological clues for the control of metastatic HCC.

Liver X Receptor Alpha Activation Inhibits Autophagy and Lipophagy in Hepatocytes by Dysregulating Autophagy-Related 4B Cysteine Peptidase and Rab-8B, Reducing Mitochondrial Fuel Oxidation.

BACKGROUND AND AIMS: Fat accumulation results from increased fat absorption and/or defective fat metabolism. Currently, the lipid-sensing nuclear receptor that controls fat utilization in hepatocytes is elusive. Liver X receptor alpha (LXRα) promotes accumulation of lipids through the induction of several lipogenic genes. However, its effect on lipid degradation is open for study. Here, we investigated the inhibitory role of LXRα in autophagy/lipophagy in hepatocytes and the underlying basis. APPROACH AND RESULTS: In LXRα knockout mice fed a high-fat diet, or cell models, LXRα activation suppressed the function of mitochondria by inhibiting autophagy/lipophagy and induced hepatic steatosis. Gene sets associated with "autophagy" were enriched in hepatic transcriptome data. Autophagy flux was markedly augmented in the LXRα knockout mouse liver and primary hepatocytes. Mechanistically, LXRα suppressed autophagy-related 4B cysteine peptidase (ATG4B) and Rab-8B, responsible for autophagosome and -lysosome formation, by inducing let-7a and microRNA (miR)-34a. Chromatin immunoprecipitation assay enabled us to find LXRα as a transcription factor of let-7a and miR-34a. Moreover, 3' untranslated region luciferase assay substantiated the direct inhibitory effects of let-7a and miR-34a on ATG4B and Rab-8B. Consistently, either LXRα activation or the let-7a/miR-34a transfection lowered mitochondrial oxygen consumption rate and mitochondrial transmembrane potential and increased fat levels. In obese animals or nonalcoholic fatty liver disease (NAFLD) patients, let-7a and miR-34a levels were elevated with simultaneous decreases in ATG4B and Rab-8B levels. CONCLUSIONS: LXRα inhibits autophagy in hepatocytes through down-regulating ATG4B and Rab-8B by transcriptionally activating microRNA let-7a-2 and microRNA 34a genes and suppresses mitochondrial biogenesis and fuel consumption. This highlights a function of LXRα that culminates in the progression of liver steatosis and steatohepatitis, and the identified targets may be applied for a therapeutic strategy in the treatment of NAFLD.

Pharmacology of Antagonism of GPCR.

Agonists are defined as the ligands that activate intracellular signaling and evoke cellular responses. Synthetic and endogenous agonists should bind specific amino acids to activate G protein-coupled receptor (GPCR). Agonists that induce maximal responses are full agonists. Partial agonists cannot induce full responses unlike full agonists. In definition, antagonists inhibit agonist-stimulated responses by binding to orthosteric or allosteric sites. Antagonists modulate agonist-induced responses and are often related with inverse agonist activity. However, the relationship between antagonists and partial agonists is complex. An antagonist behaves as a partial agonist when the constitutive activity of the GPCR is high. In contrast, a partial agonist with very weak intrinsic activity may be classified as an antagonist. Thus, antagonisms of the compounds are influenced by constitutive activity of GPCRs, intrinsic activity and differences in the binding sites of GPCRs. Since "antagonism" has been revealed to have multiple aspects and more complex than previously thought, it may be difficult to classify each compound as simply "agonist" or "antagonist" as before. In this review, we discuss the recent findings and perspectives on the pharmacology of GPCR-binding antagonists, inverse agonists, and signaling.

Hypergravity Load Modulates Acetaminophen Nephrotoxicity via Endoplasmic Reticulum Stress in Association with Hepatic microRNA-122 Expression.

Hypergravity conditions may subject the kidney to intrinsic stress and lead to hemodynamic kidney dysfunction. However, the mechanisms underlying this phenomenon remain unclear. Accumulation of unfolded proteins in the endoplasmic reticulum (i.e., ER stress) is often observed in kidney diseases. Therefore, this study investigated whether hypergravity stress alters acetaminophen-induced renal toxicity in vivo, as well as the molecular mechanisms involved in this process. C57BL/6 mice were submitted to one or three loads of +9 Gx hypergravity for 1 h with or without acetaminophen (APAP) treatment. The protein levels of cell survival markers, including pAKT and pCREB, were decreased in the kidney after acetaminophen treatment with a single hypergravity load. Additionally, the combined treatment increased kidney injury markers, serum creatinine, and Bax, Bcl2, and Kim-1 transcript levels and enhanced ER stress-related markers were further. Moreover, multiple hypergravity loads enabled mice to overcome kidney injury, as indicated by decreases in serum creatinine content and ER stress marker levels, along with increased cell viability indices. Similarly, multiple hypergravity loads plus APAP elevated miR-122 levels in the kidney, which likely originated from the liver, as the levels of primary miR-122 increased only in the liver and not the kidney. Importantly, this phenomenon may contribute to overcoming hypergravity-induced kidney injury. Taken together, our results demonstrate that APAP-exposed mice submitted to a single load of hypergravity exhibited more pronounced kidney dysfunction due to increased ER stress, which may be overcome by repetitive hypergravity loads presumably due to increased production of miR-122 in the liver. Thus, our study provides novel insights into the mechanisms by which hypergravity stress plus APAP medication induce kidney injury, which may be overcome by repeated hypergravity exposure.

UBC12-mediated SREBP-1 neddylation worsens metastatic tumor prognosis.

Activation of sterol regulatory element-binding protein 1 (SREBP-1), a master lipogenic transcription factor, is associated with cancer metabolism and metabolic disorders. Neddylation, the process of adding NEDD8 to its substrate, contributes to diverse biological processes. Here, we identified SREBP-1 as a substrate for neddylation by UBC12 and explored its impact on tumor aggressiveness. In cell-based assays, SREBP-1 neddylation prolonged SREBP-1 stability with a decrease in ubiquitination. Consequently, NEDD8 overexpression facilitated proliferation, migration, and invasion of SK-Hep1 liver tumor cells. MLN4924 (an inhibitor of the NEDD8-activating enzyme-E1) treatment or UBC12 knockdown prevented SREBP-1 neddylation and tumor cell phenotype change. This effect was corroborated in an in vivo xenograft model. In human specimens, SREBP-1, UBC12, and NEDD8 were all upregulated in hepatocellular carcinoma (HCC) compared to nontumorous regions. Moreover, SREBP-1 levels positively correlated with UBC12. In GEO database analyses, SREBP-1 levels were greater in metastatic HCC samples accompanying UBC12 upregulation. In HCC analysis, tumoral SREBP-1 and UBC12 levels discriminated overall patient survival rates. Additionally, MLN4924 treatment destabilized SREBP-1 in MDA-MB-231 breast cancer cells and in the tumor cell xenograft. SREBP-1 and UBC12 were also highly expressed in human breast cancer tissues. Moreover, most breast cancers with lymph node metastasis displayed predominant SREBP-1 and UBC12 expressions, which compromised overall patient survival rates. In summary, SREBP-1 is neddylated by UBC12, which may contribute to HCC and breast cancer aggressiveness through SREBP-1 stabilization, and these events can be intervented by MLN4924 therapy. Our findings may also provide potential reliable prognostic markers for tumor metastasis.

Nrf2-lncRNA controls cell fate by modulating p53-dependent Nrf2 activation as an miRNA sponge for Plk2 and p21cip1.

Long noncoding RNA (lncRNA) capable of controlling antioxidative capacity remains to be investigated. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a central molecule for cellular defense that increases antioxidative capacity. We identified a novel lncRNA named Nrf2-activating lncRNA (Nrf2-lncRNA) transcribed from an upstream region of the microRNA 122 gene (MIR122). Nrf2-lncRNA existed in the cytoplasm, suggestive of its function as a competing endogenous RNA [ceRNA, microRNA (miRNA) sponge]. Nrf2-lncRNA served as a ceRNA for polo-like kinase (Plk) 2 and cyclin-dependent kinase inhibitor 1 (p21cip1) through binding of miRNA 128 and miRNA 224, inducing Plk2/Nrf2/p21cip1 complexation for Nrf2 activation in the cells under p53-activating conditions (i.e., DNA damage and serum deprivation). Nrf2-lncRNA expression was suppressed with the initiation of apoptosis, being a rheostat for cell fate determination. Nrf2-lncRNA levels correlated with the recurrence-free postsurgery survival rate of patients with hepatocellular carcinoma. Collectively, Nrf2-lncRNA promotes Plk2 and p21cip1 translation by competing for specific miRNAs and activating Nrf2 under surviving conditions from oxidative stress, implying that Nrf2-lncRNA serves as a fine-tuning rheostat for cell fate decision.-Joo, M. S., Shin, S.-B., Kim, E. J., Koo, J. H., Yim, H., Kim, S. G. Nrf2-lncRNA controls cell fate by modulating p53-dependent Nrf2 activation as an miRNA sponge for Plk2 and p21cip1.

Alcohol dysregulates miR-148a in hepatocytes through FoxO1, facilitating pyroptosis via TXNIP overexpression.

OBJECTIVE: Alcoholic liver disease (ALD) is a leading cause of death among chronic liver diseases. However, its pathogenesis has not been completely established. MicroRNAs (miRNAs) are key contributors to liver diseases progression. This study investigated hepatocyte-abundant miRNAs dysregulated by ALD, its impact on hepatocyte injury and the underlying basis. DESIGN: Alcoholic hepatitis (AH) human and animal liver samples and hepatocytes were used to assess miR-148a levels. Pre-miR-148a was delivered specifically to hepatocytes in vivo using lentivirus. Immunoblottings, luciferase reporter assays, chromatin immunoprecipitation and immunofluorescence assays were carried out in cell models. RESULTS: The miRNA profile and PCR analyses enabled us to find substantial decrease of miR-148a in the liver of patients with AH. In mice subjected to Lieber-DeCarli alcohol diet or binge alcohol drinking, miR-148a levels were also markedly reduced. In cultured hepatocytes and mouse livers, alcohol exposure inhibited forkhead box protein O1 (FoxO1) expression, which correlated with miR-148a levels and significantly decreased in human AH specimens. FoxO1 was identified as a transcription factor for MIR148A transactivation. MiR-148a directly inhibited thioredoxin-interacting protein (TXNIP) expression. Consequently, treatment of hepatocytes with ethanol resulted in TXNIP overexpression, activating NLRP3 inflammasome and caspase-1-mediated pyroptosis. These events were reversed by miR-148a mimic or TXNIP small-interfering RNA transfection. Hepatocyte-specific delivery of miR-148a to mice abrogated alcohol-induced TXNIP overexpression and inflammasome activation, attenuating liver injury. CONCLUSION: Alcohol decreases miR-148a expression in hepatocytes through FoxO1, facilitating TXNIP overexpression and NLRP3 inflammasome activation, which induces hepatocyte pyroptosis. Our findings provide information on novel targets for reducing incidence and progression of ALD.

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.

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.

Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress has been implicated in a variety of diseases. Hepatic stellate cells (HSCs) contribute to the development of liver fibrosis. Information on the link between ER stress and HSC activation is scarce. We investigated the effects of ER stress in HSCs on the progression of liver fibrosis and the regulation of this process in cells and mice. METHODS: Proteins and messenger RNAs were measured in 2 sets of liver samples (n = 25 and n = 44) collected from patients with chronic hepatitis C virus infection and/or fibrosis. ER stress was induced in cells and mice using chemical agents. Lentiviral vectors were constructed to express glucose-regulated protein 78 (GRP78; also known as HSPA5) or heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) from the α-smooth muscle actin promoter and injected into C57BL/6 mice for HSC-specific gene expression. Liver tissues and HSCs were collected from mice or rats and analyzed using immunoblottings and quantitative reverse-transcription polymerase chain reaction. LX-2 cells were transfected with small interfering RNAs, microRNA mimics, or overexpression vectors. RESULTS: Hepatic ER stress was much higher in liver tissues from patients with severe vs mild fibrosis. ER stress induced fibrogenic genes in HSCs. Targeted lentiviral delivery of glucose-regulated protein 78 to HSCs in mice reduced fiber accumulation in liver. Levels of SMAD2, but not SMAD3, were increased in fibrotic liver tissues from patients or mice exposed to ER stress; small interfering RNA-mediated knockdown of SMAD2 reduced ER stress-mediated activation of HSCs. In rat HSCs, ER stress increased levels of SMAD2 messenger RNA by decreasing levels of microRNA 18a (MIR18A), an inhibitor of SMAD2 expression, rather than transactivating the SMAD2 gene. ER stress-activated PKR-like endoplasmic reticulum kinase, also known as EIF2AK3 (PERK) phosphorylated HNRNPA1, a protein required for the maturational processing of primary MIR18A, at Thr51, accelerating its degradation. Overexpression of HNRNPA1 (or its T51A mutant) in HSCs of mice inhibited liver fibrosis. Severe fibrotic liver tissues from patients had increased levels of phosphorylated PERK and reduced levels of HNRNPA1 in HSCs, compared with mild fibrotic liver tissues. CONCLUSIONS: ER stress in HSCs promotes liver fibrosis by inducing overexpression of SMAD2, via dysregulation of MIR18A; this dysregulation is mediated by PERK phosphorylation and destabilization of HNRNPA1.

Binge Alcohol Intake After Hypergravity Stress Sustainably Decreases AMPK and Transcription Factors Necessary for Hepatocyte Survival.

BACKGROUND: Binge alcohol consumption elicits mitochondrial dysfunction in hepatocytes. An understanding of the effect of ethanol (EtOH) exposure after hypergravity stress on liver function may assist in the implementation of pathophysiological countermeasures for aerospace missions. This study investigated whether a combination of hypergravity stress and binge alcohol intake has a detrimental effect on AMP-activated protein kinase (AMPK) and other molecules necessary for hepatocyte survival. METHODS: The mice were orally administered a single dose of EtOH (5 g/kg body weight, 20% EtOH) immediately after a load to +9 Gz hypergravity for 1 hour using a small animal centrifuge and sacrificed 24 hours after treatment. For the multiple-dose model, 3 consecutive daily treatments were carried out. Immunoblottings were carried out on liver homogenates. RESULTS: Binge alcohol intake in mice immediately after a 1-hour exposure to a +9 Gz hypergravity load repressed hepatic Akt and PARP-1 levels at 24 hours posttreatment. Moreover, it sustainably diminished the level of AMPKα, a key regulator of energy metabolism, as compared to each individual treatment. Similarly, the combination of alcohol and hypergravity suppressed the levels of STAT3, FOXO1/3, C/EBPß, and CREB, transcription factors necessary for cell survival. Similar changes were not detected after 3 consecutive daily combinatorial treatments, indicating that repetitive training with hypergravity loads provides hepatoprotective effects in a binge alcohol model. CONCLUSIONS: These results show that binge alcohol exposure in mice immediately following a +9 Gz hypergravity stress persistently decreased AMPKα and other key molecules required for hepatocyte survival, and these changes may be reversed by repetitive hypergravity loads.

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.