ß-Arrestin2 deficiency attenuates oxidative stress in mouse hepatic fibrosis through modulation of NOX4.

Hepatic fibrosis is a disease characterized by excessive deposition of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is responsible for most of ECM production. Oxidative stress and reactive oxygen species (ROS) may be important factors leading to liver fibrosis. NADPH oxidase 4 (NOX4) is the main source of ROS in hepatic fibrosis, but the mechanism by which NOX4 regulates oxidative stress is not fully understood. ß-Arrestin2 is a multifunctional scaffold protein that regulates receptor endocytosis, signaling and trafficking. In this study, we investigated whether ß-arrestin2 regulated oxidative stress in hepatic fibrosis. Both ß-arrestin2 knockout (Arrb2 KO) mice and wild-type mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce hepatic fibrosis. Arrb2 KO mice showed significantly attenuated liver fibrosis, decreased ROS levels and NOX4 expression, and reduced collagen levels in their livers. In vitro, NOX4 knockdown significantly inhibited ROS production, and decreased expression of alpha-smooth muscle actin in angiotensin II-stimulated human HSC cell line LX-2. Through overexpression or depletion of ß-arrestin2 in LX-2 cells, we revealed that decreased ß-arrestin2 inhibited ROS levels and NOX4 expression, and reduced collagen production; it also inhibited activation of ERK and JNK signaling pathways. These results demonstrate that ß-arrestin2 deficiency protects against liver fibrosis by downregulating ROS production through NOX4. This effect appears to be mediated by ERK and JNK signaling pathways. Thus, targeted inhibition of ß-arrestin2 might reduce oxidative stress and inhibit the progression of liver fibrosis.

Depletion of ß-arrestin 2 protects against CCl4-induced liver injury in mice.

Acute liver injury can be caused by oxidative stress within a short period and is a common pathway to many liver diseases. The liver is vulnerable to reactive oxygen species (ROS) and free radical-mediated disorders. ß-arrestin2 was initially discovered to be a negative regulator of G protein-coupled receptor signaling. Recently, ß-arrestin2 has been found to act as a multifunctional adaptor protein and play new roles in regulating intracellular signaling networks. However, the role of ß-arrestin2 in the pathogenesis of acute liver injury is unclear. In this study, we hypothesize that ß-arrestin2 regulates acute liver injury via modulation of oxidative stress. ß-arrestin2 knockout mice were used to investigate the impacts of ß-arrestin2 on carbon tetrachloride (CCl4)-induced acute liver injury and oxidative stress. Results here suggested that ß-arrestin2 deficiency decreased serum activities of aminotransferase and alleviated liver injury induced by CCl4 injection as compared with wildtype mice. ß-arrestin2 knockout mice exhibited stronger tolerance in oxidative stress compared with wild-type mice, which was demonstrated by decreased ROS level and increased superoxide dismutase (SOD) and glutathione (GSH) in the liver. Furthermore, ß-arrestin2 deficiency significantly inhibited NOX4 (a major source of ROS) expression and the activation of the extracellular regulated kinase (ERK) and, c-Jun NH2-terminal kinase (JNK) pathways. These results suggest that ß-arrestin2 deficiency protects against CCl4-induced acute liver injury through attenuating oxidative damage and decreased ERK and JNK phosphorylation.

Effects of short-term fasting on the resistance of Nile tilapia (Oreochromis niloticus) to Streptococcus agalactiae infection.

Short-term feed deprivation or fasting is commonly experienced by aquaculture fish species and may be caused by seasonal variations, production strategies, or diseases. To assess the effects of fasting on the resistance of Nile tilapia to Streptococcus agalactiae infection, vaccinated and unvaccinated fish were fasted for zero, one, three, and seven days prior to infection. The cortisol levels of both vaccinated and unvaccinated fish first decreased and then increased significantly as fasting time increased. Liver glycogen, triglycerides, and total cholesterol decreased significantly after seven days of fasting, but glucose content did not vary significantly between fish fasted for three and seven days. Hexokinase (HK) and pyruvate kinase (PK) activity levels were lowest after seven days of fasting, while phosphoenolpyruvate carboxykinase (PEPCK) activity levels varied in opposition to those of HK and PK. Serum superoxide dismutase (SOD) and catalase (CAT) activity levels first increased and then decreased as fasting time increased; SOD activity was highest after three days of fasting. Interleukin-1beta (IL-1ß) and IL-6 mRNA expression levels first increased and then decreased significantly, peaking after three days of fasting. However, suppressor of cytokine signaling-1 (SOCS-1) mRNA expression levels were in opposition to those of IL-1ß and IL-6. Specific antibody levels did not vary significantly among unvaccinated fish fasted for different periods. Although specific antibody level first increased and then decreased in the vaccinated fish as fasting duration increased, there were no significant differences in the survival rates of fasted vaccinated fish after challenge with S. agalactiae. The final survival rates of vaccinated fish fasted for zero, one, three, and seven days were 86.67 ±â€¯5.44%, 80.00 ±â€¯3.14%, 88.89 ±â€¯6.28%, and 84.44 ±â€¯8.32%, respectively. Among the unvaccinated fish, the survival rate was highest (35.56 ±â€¯3.14%) in the fish fasted for three days and lowest (6.67 ±â€¯3.14%) in the fish fasted for seven days. Therefore, our results indicated that short-term fasting (three days) prior to an infection might increase the resistance of unvaccinated Nile tilapia to S. agalactiae.

Transcriptome analysis provides insights into molecular immune mechanisms of rabbitfish, Siganus oramin against Cryptocaryon irritans infection.

The rabbitfish Siganus oramin is resistant to the ciliate parasite Cryptocaryon irritans. L-amino acid oxidase (LAAO) protein from rabbitfish can kill C. irritans in vitro, however, other immune defence mechanisms against C. irritans remains unknown. Here, we generated transcriptomes of rabbitfish skin at 12 h post infection (PI) by C. irritans. The transcriptomes contained 238, 504, 124 clean reads were obtained and then assembled into 258,869 unigenes with an average length of 621 bp and an N50 of 833 bp. Among them, we obtained 418 differentially expressed genes (DEGs) in the skin of rabbitfish under C. irritans infection and control conditions, including 336 significantly up-regulated genes and 82 significantly down-regulated genes. Seven immune-related categories with 32 differentially expressed immune genes were obtained using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. DEGs included innate immune molecules, such as LAAO, antimicrobial peptide, lysozyme g, as well as complement components, chemokines and chemokine receptors, NOD-like receptor/Toll-like receptor signaling pathway molecules, antigen processing and T/B cell activation and proliferation molecules. We further validated the expression results of nine immune-related DEGs using quantitative real-time PCR. This study provides new insights into the early immune response of a host that is resistant to C. irritans.

Emerging Roles of G Protein-Coupled Receptors in Hepatocellular Carcinoma.

Among a great variety of cell surface receptors, the largest superfamily is G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors. GPCRs can modulate diverse signal-transduction pathways through G protein-dependent or independent pathways which involve β-arrestins, G protein receptor kinases (GRKs), ion channels, or Src kinases under physiological and pathological conditions. Recent studies have revealed the crucial role of GPCRs in the tumorigenesis and the development of cancer metastasis. We will sum up the functions of GPCRs—particularly those coupled to chemokines, prostaglandin, lysophosphatidic acid, endothelin, catecholamine, and angiotensin—in the proliferation, invasion, metastasis, and angiogenesis of hepatoma cells and the development of hepatocellular carcinoma (HCC) in this review. We also highlight the potential avenues of GPCR-based therapeutics for HCC.

A homologue of vitamin K epoxide reductase in Solanum lycopersicum is involved in resistance to osmotic stress.

Vitamin K epoxide reductase (VKOR) exists widely in the chloroplasts of higher plants and plays important roles in redox regulation. However, investigations of plant VKOR function have primarily focused on VKOR from Arabidopsis, and knowledge concerning this function is therefore quite limited. In this study, Solanum lycopersicum was used to study the function of VKOR. Transcription level analysis revealed that SlVKOR (S. lycopersicum VKOR) expression was upregulated by salt or drought stress. To further investigate the function of SlVKOR in plants, we generated sense and antisense transgenic S. lycopersicum homozygotes at T2 generation plants. Compared with wild-type (WT) plants, the leaf disks of the SlVKOR overexpression plants retained a much higher percentage of chlorophyll after salt or drought treatment, whereas the antisense transgenic plants displayed an opposite response. The overexpressed plants displayed lower levels of H2O2 and superoxide anion radical (O2(·-)) than WT plants, whereas antisense plants accumulated considerably more H2O2 and O2(·-). The activities of reactive oxygen scavenger enzymes, including superoxide dismutase, peroxidase, ascorbate peroxidase and catalase, were consistent with the accumulation of reactive oxygen species. Based on these results, we suggest that SlVKOR is involved in resistance to salt or drought stress.

Thylakoid membrane oxidoreductase LTO1/AtVKOR is involved in ABA-mediated response to osmotic stress in Arabidopsis.

Arabidopsis lumen thiol oxidoreductase 1 (LTO1) - the At4g35760 gene product - was previously found to be related to reactive oxygen species (ROS) accumulation. Here, we show that ROS accumulated in a mutant Arabidopsis line (lto1-2, mutant of LTO1/AtVKOR) under osmotic stress at a higher level than that observed in wild-type and transgenic complemented plants of the lto1-2 mutant (lto1-2C, transgenic complemented plants of lto1-2). Because ROS accumulation in osmotic stress is triggered by abscisic acid (ABA), an ABA-responsive gene, Annexin 1 (AnnAt1), was selected to study the response. Osmotic stress or exogenous ABA can significantly upregulate the transcription of AnnAt1 in wild-type and lto1-2C plants. Only a slight change in the transcriptional abundance of AnnAt1 was observed under osmotic stress in the lto1-2 mutant, but exogenous ABA application could increase the expression of AnnAt1, which suggested that exogenous ABA had a partial complementation role. Because the transcription of AnnAt1 is regulated by ABRE (ABA-responsive elements) binding proteins (AREBs)/ABRE binding factors (ABFs), the expression of AREBs/ABFs was also analyzed. The transcription of AREBs/ABFs in the lto1-2 mutant was not induced by osmotic stress but was significantly upregulated by exogenous ABA, which significantly differs from the wild-type and lto1-2C plant responses. Similarly, the expression of another ABA-responsive gene, RD29B (responsive to desiccation stress gene 29B), in the lto1-2 mutant was also upregulated by exogenous ABA. The partial complementation of mutants by ABA indicated that the ABA signal transduction pathway was not significantly affected in the lto1-2 mutant. Taken together, these results suggest that LTO1 is involved in ABA-mediated response to osmotic stress, possibly by affecting the biosynthesis of endogenous ABA.

Identification and characterization of SlVKOR, a disulfide bond formation protein from Solanum lycopersicum, and bioinformatic analysis of plant VKORs.

Homologs of vitamin K epoxide reductase (VKOR) exist widely in plants. However, only VKOR of Arabidopsis thaliana has been the subject of many studies to date. In the present study, the coding region of a VKOR from Solanum lycopersicum (JF951971 in GenBank) was cloned; it contained a membrane domain (VKOR domain) and an additional soluble thioredoxin-like (Trx-like) domain. Bioinformatic analysis showed that the first 47 amino acids in the N-terminus should act as a transit peptide targeting the protein to the chloroplast. Western blot demonstrated that the protein is localized in thylakoid membrane with the Trx-like domain facing the lumen. Modeling of three-dimensional structure showed that SlVKOR has a similar conformation with Arabidopsis and cyanobacterial VKORs, with five transmembrane segments in the VKOR domain and a typical Trx-like domain in the lumen. Functional assay showed that the full-length of SlVKOR with Trx-like domain without the transit peptide could catalyze the formation of disulfide bonds. Similar transit peptides at the N-terminus commonly exist in plant VKORs, most of them targeting to chloroplast according to prediction. Comparison of sequences and structures from different plants indicated that all plant VKORs possess two domains, a transmembrane VKOR domain and a soluble Trx-like domain, each having four conservative cysteines. The cysteines were predicted to be related to the function of catalyzing the formation of disulfide bonds.

ß-arrestin2 regulating ß2-adrenergic receptor signaling in hepatic stellate cells contributes to hepatocellular carcinoma progression.

Background: ß-arrestin2 and ß2-adrenergic receptor (ß2-AR) have important roles in malignant tumors, the present study aims to investigate the role of activated ß2-AR in hepatic stellate cells (HSCs) during hepatocellular carcinoma (HCC) progression and the regulatory effect of ß-arrestin2. Methods: Immunofluorescence and Western blot were used to detect the expression of ß-arrestin2 and ß2-AR in HSCs of liver tissues from human HCC samples and diethylnitrosamine (DEN)-induced HCC model mice. We next used ß-arrestin2-/- mice to demonstrate the regulatory role of ß-arrestin2 in DEN mice. The subsets of T cells were quantified by flow cytometry. MTT and wound healing assay were applied to detect the proliferation and migration of cells. Co-immunoprecipitation assay was used to detect the link of ß-arrestin2 and ß2-AR in HSCs. Effect of ß-arrestin2 overexpression on ß2-AR downstream signaling pathway was verified by Western blot. The secretion of CCL2 was detected by ELISA. Results: The expression of ß2-AR was significantly increased, while ß-arrestin2 was decreased in HSCs of HCC tissues. And ß-arrestin2 deficiency exacerbates DEN-induced HCC accompanied with increased ß2-AR expression. The results of flow cytometry showed that the percentage of activated T cells decreased gradually after DEN injection. ß-arrestin2 knockout down-regulated the ratio of activated T cells. In vitro, selective activation of ß2-AR in HSCs promoted the proliferation and migration of HCC cells. ß-arrestin2 overexpression enhanced co-immunoprecipitation of ß-arrestin2 and ß2-AR in activated HSCs, and decreased its downstream Akt phosphorylation. Akt inhibitor decreased secretion of CCL2 in activated HSCs. Conclusion: Our study demonstrated that ß2-AR activation in HSCs induces the proliferation and migration of HCC cells may be through Akt signaling, and this effect appears to be regulated by ß-arrestin2.

GRK2 Suppresses Hepatocellular Carcinoma Metastasis and Invasion Through Down-Regulation of Prostaglandin E Receptor 2.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive form of human liver cancer and the fifth most common malignancy worldwide. Novel effective treatment strategies for HCC are urgently in clinical because of its poor response to conventional therapies. G protein-coupled receptor kinases (GRKs), including GRK2 and GRK3, are known that involves in various essential cellular processes and regulates numerous signaling pathways. However, the role of GRK2/3 in invasion and metastasis of HCC still remains unclear. MATERIALS AND METHODS: Immunohistochemistry, Western blot, laser confocal microscopy and qRT-PCR were used to detect the expression of GRK2/3 and EP2 in liver tissues of HCC patients and DEN-induced HCC mice. Wound healing and transwell assay were applied to measure the migration and invasion of HCC cells after transfected with GRK2 siRNA. The downstream pathway of Akt and ERK was verified by Western blot. RESULTS: The expression of GRK2 was significantly decreased, while GRK3 was not significantly changed in HCC tissues compared with noncancerous tissues of HCC patients. Moreover, GRK2 expression was reduced during liver tumorigenesis in diethylnitrosamine-induced liver tumor model. In addition, our in vitro study showed that GRK2 expression was gradually decreased with increasing HCC cell line metastatic potential, and GRK2 knockdown significantly promoted the migration and invasion of HCC cells. Furthermore, low GRK2 expression was associated with increased expression of EP2 receptor translocation to HCC cell membrane, and the activation of Akt pathway. CONCLUSION: These data suggest that GRK2 inhibits HCC metastasis and invasion may be through regulating EP2 receptor translocation, and this effect appears to be mediated by Akt pathway.

ß-arrestin2 deficiency protects against hepatic fibrosis in mice and prevents synthesis of extracellular matrix.

Hepatic fibrosis is a disease of the wound-healing response following chronic liver injury, and activated hepatic stellate cells (HSCs) play a crucial role in the progression of hepatic fibrosis. ß-arrestin2 functions as a multiprotein scaffold to coordinate complex signal transduction networks. Although ß-arrestin2 transduces diverse signals in cells, little is known about its involvement in the regulation of liver fibrosis. Our current study utilized a porcine serum-induced liver fibrosis model and found increased expression of ß-arrestin2 in hepatic tissues with the progression of hepatic fibrosis, which was positively correlated with collagen levels. Furthermore, changes in human fibrotic samples were also observed. We next used ß-arrestin2-/- mice to demonstrate that ß-arrestin2 deficiency ameliorates CCl4-induced liver fibrosis and decreases collagen deposition. The in vitro depletion and overexpression experiments showed that decreased ß-arrestin2 inhibited HSCs collagen production and elevated TßRIII expression, thus downregulating the TGF-ß1 pathway components Smad2, Smad3 and Akt. These findings suggest that ß-arrestin2 deficiency ameliorates liver fibrosis in mice, and ß-arrestin2 may be a potential treatment target in hepatic fibrosis.

From helix to helical pores: solid-state crystalline conversions triggered by gas-solid reactions.

Two one-dimensional (1D) helical coordination polymers, CuBIm-Cl and CuBIm-Br (BIm = 1,2-bis((5H-imidazol-4-yl)-methylene)hydrazine), are constructed from Cu2+ ions, halides and mono-deprotonated BIm. Solid-state crystalline conversions of CuBIm-Cl and CuBIm-Br to a gyroidal metal-organic framework (MOF) STU-3 which is composed of Cu ions and Bim ligands are observed through a gas-solid reaction, where the gas is methylamine.

The conservative cysteines in transmembrane domain of AtVKOR/LTO1 are critical for photosynthetic growth and photosystem II activity in Arabidopsis.

Thylakoid protein vitamin K epoxide reductase (AtVKOR/LTO1) is involved in oxidoreduction. The deficiency of this compound causes pleiotropic defects in Arabidopsis thaliana, such as severely stunted growth, smaller sized leaves, and delay of flowering. Transgenic complementation of wild-type AtVKOR (VKORWT) to vkor mutant lines ultimately demonstrates that the phenotype changes are due to this gene. However, whether AtVKOR functions in Arabidopsis through its protein oxidoreduction is unknown. To further study the redox-active sites of AtVKOR in vivo, a series of plasmids containing cysteine-mutant VKORs were constructed and transformed into vkor deficient lines. Compared with transgenic AtVKORWT plants, the size of the transgenic plants with a single conservative cysteine mutation (VKORC109A, VKORC116A, VKORC195A, and VKORC198A) were smaller, and two double-cysteine mutations (VKORC109AC116A and VKORC195AC198A) showed significantly stunted growth, similar with the vkor mutant line. However, mutations of two non-conservative cysteines (VKORC46A and VKORC230A) displayed little obvious changes in the phenotypes of Arabidopsis. Consistently, the maximum and actual efficiency of photosystem II (PSII) in double-cysteine mutation plants decreased significantly to the level similar to that of the vkor mutant line both under normal growth light and high light. A significantly decreased amount of D1 protein and increased accumulation of reactive oxygen species were observed in two double-cysteine mutations under high light. All of the results above indicated that the conservative cysteines in transmembrane domains were the functional sites of AtVKOR in Arabidopsis and that the oxidoreductase activities of AtVKOR were directly related to the autotrophic photosynthetic growth and PSII activity of Arabidopsis thaliana.

Key amino acids of arabidopsis VKOR in the activity of phylloquinone reduction and disulfide bond formation.

Many proteins in chloroplast are regulated through the disulfide bond/thiol transformation to realize their activities. A homologue of VKOR (Vitamin K epoxide reductase) in Arabidopsis chloroplast is found to catalyze the disulfide bond formation in vivo and to mediate the specific phylloquinone reduction in vitro. It is also called LTO1 (Lumen Thiol Oxidoreductase 1). Investigations about functions and essential amino acid residues of AtVKOR have important theoretical significance to clarify the chloroplast redox regulation mechanism. In this study, several amino acids in the VKOR domain of AtVKOR were identified to be involved in binding of phylloquinone. Site-directed mutagenesis was used to study the function of these positions. The results suggested that residues Ser77, Leu87, Phe137 and Leu141 were quite important in the binding and catalyzing the reduction of phylloquinone. These residues were also involved in the electron transferring and disulfide bond formation of substrate proteins by motility assays in vivo, suggesting that the binding of phylloquinone not only affected the delivery of electrons to phylloquinone but also affected the whole electron transfer process. The conserved cysteines in the AtVKOR domain also played critical roles in phylloquinone reduction. When each of the four conserved cysteines was mutated to alanine, the mutants lost reduction activity entirely, suggesting that the four conserved cysteines played crucial roles in the electron transfer process.

The chloroplast protein LTO1/AtVKOR is involved in the xanthophyll cycle and the acceleration of D1 protein degradation.

The thylakoid protein LTO1/AtVKOR-DsbA is recently found to be an oxidoreductase involved in disulfide bond formation and the assembly of photosystem II (PSII) in Arabidopsis thaliana. In this study, experimental evidence showed that LTO1 deficiency caused severe photoinhibition which was related to the xanthophyll cycle and D1 protein degradation. The lto1-2 mutant was more sensitive to intense irradiance than wild type. When treated with different concentrations of dithiothreitol (DTT), an inhibitor of violaxanthin de-epoxidase (VDE) in the xanthophyll cycle, there was a larger reduction in NPQ in the wild type than in the lto1-2 mutant under high irradiance, indicating that lto1-2 had a lower sensitivity to DTT gradients than did the wild type. Zeaxanthin in the xanthophyll cycle, which participates in the thermal dissipation of excess absorbed light energy, was much less active in lto1-2 than in the wild type under intense light levels, and the de-epoxidation state of the xanthophyll cycle was consistent with the susceptibility of NPQ. Together these observations indicated that aggravated photoinhibition in lto1-2 was related to a reduction in xanthophyll cycle-associated energy dissipation. When D1 protein synthesis was suppressed by an inhibitor of chloroplast protein synthesis (streptomycin sulfate), the levels of D1 protein decreased more in the lto1-2 mutant than in the wild type when exposed to intense light levels, implying that a deficiency in LTO1 accelerated the degradation of D1 and thus affected D1 turnover. Transgenic complementation of plants with lto1-2 ultimately allowed for the recovery of the photoinhibition properties of leaves.

Identification of potential targets for thylakoid oxidoreductase AtVKOR/LTO1 in chloroplasts.

The Arabidopsis thylakoid membrane bimodular oxidoreductase, AtVKOR, could catalyze disulfide bond formation, and its direct functional domain (thioredoxin-like domain) is located in the thylakoid lumen according to the topological structure. Many proteins have one or several disulfide bonds in the thylakoid lumen, including photosynthetic chain components. A yeast two-hybrid assay was used to identify potential targets for the AtVKOR, and a Trx-like domain was constructed into a BD vector as bait. Twenty-two thylakoid lumenal proteins with disulfides were selected. The cDNAs encoding these proteins were constructed into an AD vector. Eight proteins were identified from the hybrid results to interact with AtVKOR, including HCF164, cytochrome c6A, violaxanthin deepoxidase, embryo sac development arrest 3 protein (EDA3), two members pentapeptide repeat proteins (TL17 and TL20.3), and two FK-506 binding proteins (FKBP13 and FKBP20-2). The BIACORE system was used to demonstrate that the recombinant HCF164 and Trx-like domain of AtVKOR could interact directly in vitro. The KD value for binding HCF164 to AtVKOR was calculated as 2.5×10(-6) M. These results suggest that AtVKOR can interact with partial thylakoid lumenal proteins and indicates AtVKOR plays an important role in regulating the thylakoid lumen redox.