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MicroRNAs (miRNAs or miRs) have been found to participate in the development and malignant progression of human cancers by negatively mediating the expression of their target genes. Recently, miR‑33b has been reported to be involved in multiple types of human cancer, including hepatocellular carcinoma (HCC). However, the underlying regulatory mechanisms of miR‑33b in HCC cell growth and metastasis remain largely unclear. In the present study, RT-qPCR revealed that miR‑33b was significantly downregulated in HCC tissues compared to their matched adjacent normal tissues. Moreover, the miR‑33b level was significantly lower in advanced-stage HCC (stages T3-T4) compared to early-stage HCC (stages T1-T2). Furthermore, it was also downregulated in the HCC cell lines, LH86, HepG2, LMH and PLHC-1, when compared with the THLE-3 normal human liver cells. We further demonstrated that the overexpression of miR‑33b led to a significant decrease in the proliferation, migration and invasion of HepG2 and LH86 cells. Luciferase reporter assay identified Sal-like protein 4 (SALL4) as a target gene of miR‑33b, and its protein expression was negatively regulated by miR‑33b in HepG2 and LH86 cells. Moreover, the restoration of SALL4 expression markedly reversed the inhibitory effect of miR‑33b overexpression on the proliferation, migration and invasion of HepG2 and LH86 cells, indicating that SALL4 is involved in miR‑33b-mediated malignant phenotypes of HCC cells. Furthermore, we found that SALL4 was significantly upregulated in HCC tissues compared to their matched adjacent normal tissues, and its increased expression was significantly associated with the advanced malignancy of HCC. Moreover, SALL4 was also upregulated in HCC cell lines compared to the THLE-3 normal human liver cells. Finally, we found that the SALL4 expression inversely correlated with the miR‑33b level in HCC tissues. On the whole, the findings of our study demonstrate that miR‑33b suppresses the proliferation and metastasis of HCC cells through the inhibition of SALL4 expression. Therefore, miR‑33b/SALL4 may become a potential therapeutic target for the treatment of HCC.

Non-alcoholic steatohepatitis (NASH) has no approved therapy. The farnesoid X nuclear receptor (FXR) agonist obeticholic acid (OCA) has shown promise as a drug for NASH, but can adversely affect plasma lipid profiles. Therefore, the present study aimed to investigate the effects and underlying mechanisms of OCA in combination with simvastatin (SIM) in a high-fat diet (HFD)-induced model of NASH. C57BL/6J mice were fed with a HFD for 16 weeks to establish the NASH model. The mice were randomly divided into the following five groups: HFD, HFD + OCA, HFD + SIM, HFD + OCA + SIM and control. After 16 weeks, the mice were sacrificed under anesthesia. The ratios of liver weight to body weight (Lw/Bw) and of abdominal adipose tissue weight to body weight were calculated. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol, triglycerides and low-density lipoprotein were measured. Liver sections were stained with hematoxylin and eosin. The protein levels of FXR, small heterodimeric partner (SHP) and cytochrome P450 family 7 subfamily A member 1 (CYP7A1) in the liver were detected by western blotting, while the mRNA levels of FXR, SHP, CYP7A1, bile salt export pump, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), sterol regulatory element binding protein-1 (SREBP1) and fatty acid synthase (FASN) were examined by reverse transcription-quantitative polymerase chain reaction. The administration of OCA with or without SIM reduced the liver inflammation score compared with those of the HFD and HFD + SIM groups, with no significant difference between the HFD + OCA and HFD + OCA + SIM groups. The steatosis score followed similar trends to the inflammation score. In HFD-fed mice, OCA combined with SIM prevented body weight gain compared with that in HFD and HFD + OCA groups, and reduced the Lw/Bw ratio compared with that in the HFD and HFD + SIM groups. In addition to preventing HFD-induced increases of ALT and AST, the combination of OCA and SIM reduced the mRNA levels of IL-6, TNF-α, SREBP1 and FASN. On the basis of these results, it may be concluded that the strategy of combining OCA with SIM represents an effective pharmacotherapy for NASH.

Aims To explore the potential regulatory mechanism of differentially expressed mRNAs in Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Main methods Patients with HCV-related HCC and age- and gender-matched healthy subjects were enrolled. Differentially expressed mRNAs in the plasma were detected by digital gene expression (DGE) profile analysis. HepG2 and SMMC7721 cells stably transfected with HCV-core protein and the control plasmid were established. And small interfering RNA (siRNA) was used to knockdown the target gene in HCV core-expressing HCC cell lines. mRNA expression was determined by qRT-PCR. Protein expression was measured by Western blot and immunohistochemistry staining. Key findings DGE profile data showed aberrant mRNA expression contributed to the progression of HCV-HCC, and clusterin (CLU), which was significantly highly expressed, was chosen as a candidate gene. Further evidence showed CLU was highly expressed in tumor tissues of HCV-HCC patients and HCV core-expressing HCC cell lines, accompanied with enhanced autophagy and upregulation of pro-autophagy genes. And knockdown of CLU in HCC cell lines suppressed cell autophagy, which was indicated by decreased expression of autophagy marker light chain 3B (LC3B) ІІ/І ratio, and downregulated pro-autophagy genes like Beclin1, autophagy-related protein 7 (Atg7) and Lamp2. On the other hand, anti-autophagy genes or regulators, including p62 and phosphorylated mammalian target of rapamycin (p-mTOR), were notably upregulated. Significance CLU could promote the progression of HCV-related HCC by regulating autophagy, which might be a potential therapeutic target of HCV-HCC.

MicroRNA (miRNA) play a pivotal role in the development of liver fibrosis. However, the functions of miRNA in hepatitis C virus (HCV)-related liver fibrosis remain unclear. In this study, we systematically analyzed the microarray data of the serum miRNA in patients with HCV-induced hepatic fibrosis. Among 41 dysregulated miRNA, miR-1273g-3p was the most significantly upregulated miRNA and correlated with the stage of liver fibrosis. Overexpression of miR-1273g-3p could inhibit translation of PTEN, increase the expression of α-SMA, Col1A1, and reduce apoptosis in HSCs. Hence, we conclude that miR-1273g-3p might affect the activation and apoptosis of HSCs by directly targeting PTEN in HCV-related liver fibrosis.

Objective To investigate the regulation mechanism of T cell immunoglobulin and mucin domain-3 (Tim-3) combined with toll-like receptor 3 (TLR3) or TLR4 on antiviral immune and inflammatory response in patients with chronic hepatitis C virus (HCV) infection. Methods Patients with chronic HCV infection and healthy control subjects were recruited. Patients received interferon (IFN)-α based therapy. Plasma galectin-9 (Gal-9) was quantitated. Peripheral blood mononuclear cells (PBMCs) were cultured with TLR3 or TLR4 agonists, alone or in combination with Tim-3 antagonist. Levels of IFN-α, TNF-α, and 2′-5′ oligoadenylate synthetase (2′-5′OAS), myxovirus resistance protein A (MxA) and suppressor of cytokine 1 (SOCS1) RNA in PBMC cultures were evaluated. Results Plasma Gal-9 levels were increased in patients ( n = 52) compared with controls ( n = 20) and significantly declined at treatment week 12 and 24 weeks post-treatment. IFN-α, 2′-5′OAS, MxA, TNF-α and SOCS1 were upregulated by TLR3 and TLR4 agonists. TNF-α and SOCS1 levels were suppressed by the addition of Tim-3 antagonist. Conclusions Tim-3 blockade in combination with TLR activation induces the expression of antiviral molecules without a significant increase in TNF-α or SOCS1.

Abscisic acid (ABA) plays an essential role in seed germination. In this study, we demonstrate that one SNF1-RELATED PROTEIN KINASE3-type protein kinase, SOS2-LIKE PROTEIN KINASE5 (PKS5), is involved in ABA signal transduction via the phosphorylation of an interacting protein, ABSCISIC ACID-INSENSITIVE5 (ABI5). We found that pks5-3 and pks5-4, two previously identified PKS5 superactive kinase mutants with point mutations in the PKS5 FISL/NAF (a conserved peptide that is necessary for interaction with SOS3 or SOS3-LIKE CALCIUM BINDING PROTEINs) motif and the kinase domain, respectively, are hypersensitive to ABA during seed germination. PKS5 was found to interact with ABI5 in yeast (Saccharomyces cerevisiae), and this interaction was further confirmed in planta using bimolecular fluorescence complementation. Genetic studies revealed that ABI5 is epistatic to PKS5. PKS5 phosphorylates a serine (Ser) residue at position 42 in ABI5 and regulates ABA-responsive gene expression. This phosphorylation was induced by ABA in vivo and transactivated ABI5. Expression of ABI5, in which Ser-42 was mutated to alanine, could not fully rescue the ABA-insensitive phenotypes of the abi5-8 and pks5-4abi5-8 mutants. In contrast, mutating Ser-42 to aspartate rescued the ABA insensitivity of these mutants. These data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for the ABA regulation of seed germination and gene expression in Arabidopsis (Arabidopsis thaliana).

Liver cirrhosis is recognized as being the consequence of immune-mediated hepatocyte damage and repair processes. However, the regulation of these immune responses underlying liver cirrhosis has not been elucidated. In this study, we used GEO datasets and bioinformatics methods to established coding and non-coding gene regulatory networks including transcription factor-/lncRNA-microRNA-mRNA, and competing endogenous RNA interaction networks. Our results identified 2224 mRNAs, 70 lncRNAs and 46 microRNAs were differentially expressed in liver cirrhosis. The transcription factor -/lncRNA- microRNA-mRNA network we uncovered that results in immune-mediated liver cirrhosis is comprised of 5 core microRNAs (e.g., miR-203; miR-219-5p), 3 transcription factors (i.e., FOXP3, ETS1 and FOS) and 7 lncRNAs (e.g., ENTS00000671336, ENST00000575137). The competing endogenous RNA interaction network we identified includes a complex immune response regulatory subnetwork that controls the entire liver cirrhosis network. Additionally, we found 10 overlapping GO terms shared by both liver cirrhosis and hepatocellular carcinoma including "immune response" as well. Interestingly, the overlapping differentially expressed genes in liver cirrhosis and hepatocellular carcinoma were enriched in immune response-related functional terms. In summary, a complex gene regulatory network underlying immune response processes may play an important role in the development and progression of liver cirrhosis, and its development into hepatocellular carcinoma.

Objective(s) Long noncoding RNAs (lncRNAs)-activated by transforming growth factor beta (lncRNA-ATB) is known to be involved in the invasion of hepatocellular carcinoma by regulating target genes of miR-200a. However, the role and molecular mechanisms of lncRNA-ATB/miR-200a in HCV-related liver fibrosis remains unclear. In this study, we examined the expression of lncRNA-ATB/miR-200a, and their target gene β-Catenin in liver tissues of HCV patients and hepatic stellate cells (HSCs) to elucidate the possible role of lncRNA-ATB/miR-200a axis in HSC activation and development of liver fibrosis. Materials and methods Liver tissues were obtained by biopsy or surgery from eighteen HCV patients with severe liver fibrosis and six healthy subjects (control). Conditioned media (CM) from cultured HepG2-CORE cells (HepG2 cells stably expressing HCV core protein) were used to treat LX-2 cells. The binding sites between lncRNA-ATB/miR-200a and β-catenin were predicted and then verified by a dual luciferase reporter assay. The effect of lncRNA-ATB/miR-200a/β-catenin on HSC activation was assessed by examining the expression of alpha-smooth muscle actin (α-SMA) and collagen type 1 alpha 1 (Col1A1) in HSCs. Further, the regulatory role of lncRNA-ATB on HSC activation and miR-200a/β-catenin expression was assessed by using siRNA-mediated knockdown of lncRNA-ATB. Results LncRNA-ATB was up-regulated in fibrotic liver tissues and activated LX-2 cells treated with CM from HepG2-CORE cells. Dual luciferase reporter assays confirmed that lncRNA-ATB contained common binding sites for miR-200a and β-catenin. Decreased expression of miR-200a and increased expression of β-catenin were observed in liver tissues of patients with HCV-related hepatic fibrosis and activated HSCs. Knockdown of lncRNA-ATB could down-regulate β-catenin expression by up-regulating the endogenous miR-200a and suppress the activation of LX-2 cells. Conclusion LncRNA-ATB/miR-200a/β-catenin regulatory axis likely contributed to the development of liver fibrosis in HCV patients. Knockdown of lncRNA-ATB might be a novel therapeutic target for HCV-related liver fibrosis.

Interleukin-23 (IL-23) and its downstream factor IL-17 are the key cytokines involved in immune and inflammatory response in chronic liver diseases. This study aimed to investigate the role and molecular mechanisms of the IL-23/Th17 axis in chronic hepatitis C virus (HCV) infection, and the efficacy of IL-23/Th17 modulation in response to anti-HCV therapy. Sixty-six HCV-infected patients and 20 healthy controls were enrolled. The patients received PegIFNa-2a and ribavirin therapy for at least 48 weeks. The plasma level of IL-23 and the number of IL-17A-, IFN-γ-, and IL-21-producing peripheral blood mononuclear cells (PBMCs) at baseline and 12, 24, and 48 weeks following treatment were determined. The mRNA level of Th17 immune-associated molecules in PBMCs was evaluated by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) following treatment with IL-23 agonist or antagonist. Our data showed that, compared to healthy controls, HCV-infected patients had an increased plasma level of IL-23 and increased frequencies of IL-17A- and IFN-γ-producing PBMCs, whereas the HCV patients exhibited a reduced number of IL-21-producing PBMCs. However, the baseline frequencies of IL-21-producing PBMCs were markedly higher in HCV patients who achieved rapid virological response (RVR) than those without RVR. Additionally, the mRNA expressions of IL-21, IFN-γ, myxovirus resistance protein A (MxA), and suppressor of cytokine signaling 3 (SOCS3) were significantly upregulated in PBMCs, while FoxP3 expression was suppressed by IL-23 agonist. Thus, the IL-23/Th17 axis plays an important role in development of chronic HCV infection and antiviral response. IL-23 may enhance the antiviral activity of interferon-based therapy by modulating the expression of Th17 cells-associated molecules in HCV-infected patients.

Nonalcoholic fibrosing steatohepatitis is a uniform process throughout nonalcoholic fatty liver disease (NAFLD). MicroRNAs (miRNAs) have been suggested to modulate cellular processes in liver diseases. However, the functional role of miRNAs in nonalcoholic fibrosing steatohepatitis is largely unclear. In this study, we systematically analyzed the hepatic miRNAs by microarray analysis in nonalcoholic fibrosing steatohepatitis in C57BL/6J mice induced by methionine-choline deficient (MCD) diet. We identified 19 up-regulated and 18 down-regulated miRNAs in liver with fibrosis. Among these dysregulated miRNAs, miR-146a-5p was the most significant down-regulated miRNA. Luciferase activity assay confirmed that Wnt1 and Wnt5a were both the target genes of miR-146a-5p. Hepatic miR-146a-5p was down-regulated in fibrosing steatohepatitis, but its target genes Wnt1 and Wnt5a and their consequent effectors α-SMA and Col-1 were significantly up-regulated. In addition, miR-146a-5p was downregulated, whilst Wnt1 and Wnt5a were up-regulated in the activated primary hepatic stellate cells (HSCs) compared to the quiescent primary HSCs. Overexpression of miR-146a-5p in HSCs inhibited HSC activation and proliferation, which concomitant with the decreased expressions of Wnt1, Wnt5a, α-SMA and Col-1. In conclusion, miR-146a-5p suppresses activation and proliferation of HSCs in the progress of nonalcoholic fibrosing steatohepatitis through targeting Wnt1 and Wnt5a and consequent effectors α-SMA and Col-1.

We have taken advantage of the close structural relationship between two C-terminal motors, KIFC5A and KIFC1, to examine the sequence requirements for targeting of these two motors within the cell. Although KIFC5A and KIFC1 are almost identical in their motor and stalk domains, they differ in well-defined regions of their tail domains. Specific antisera to these motors were used to determine their localization to distinct subcellular compartments, the spindle for KIFC5A or membranous organelles for KIFC1. In addition to defining the intracellular localization of KIFC1, the reactivity of the KIFC1 antibody demonstrates that this motor contains a frame shift with respect to KIFC5A and is likely the product of a separate gene. The divergent tail domains of these motors are predicted to harbor specific information that directs them to their correct intracellular targets. In order to define the sequences responsible for the differential localization of these two motors, GFP was fused to motors with various tail deletions and their localization visualized after transfection. We were able to identify distinct sequences in each motor responsible for its unique cellular localization. The KIFC5A tail contains a 43 amino acid sequence with both nuclear localization and microtubule binding activity while KIFC1 contains a 19 amino acid sequence sufficient to target this motor to membrane-bounded organelles.

Nonalcoholic fibrosing steatohepatitis is a uniform process that occurs throughout nonalcoholic fatty liver disease (NAFLD). MicroRNAs (miRNAs) have been shown to be involved in the biological processes, but the role and molecular mechanism of miRNAs in NAFLD are not entirely clear. In this study, we observed a significant reduction in the expression of miR-130a-3p in livers of a mouse model with fibrosis induced by a methionine–choline-deficient diet, of NAFLD patients, and in activated hepatic stellate cells (HSCs). A dual-luciferase activity assay confirmed that transforming growth factor-beta receptors (TGFBRs) 1 and 2 were both the target genes of miR-130a-3p. The hepatic expression of TGFBR1 and TGFBR2 was significantly increased. Moreover, the overexpression of miR-130a-3p in HSCs inhibited HSC activation and proliferation, concomitant with the decreased expression of TGFBR1, TGFBR2, Smad2, Smad3, matrix metalloproteinase-2 (MMP-2), MMP-9, type I collagen (Col-1), and Col-4. In addition, the overexpression of miR-130a-3p promoted HSC apoptosis by inducing the expression of caspase-dependent apoptosis genes. Transfection with si-TGFBR1 and si-TGFBR2 revealed effects on HSC function that were consistent with those of miR-130a-3p. TGFBR1 and TGFBR2 rescued the miR-130a-3p-mediated reductions in the mRNA and protein expression levels of Smad2, Smad3, Col-1, and Col-4. In conclusion, our findings suggest that miR-130a-3p might play a critical role in negatively regulating HSC activation and proliferation in the progression of nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2 via the TGF-β/SMAD signaling pathway.

Microfilament and Ca2+ dynamics play important roles in stress signaling in plants. Through genetic screening of Arabidopsis thaliana mutants that are defective in stress-induced increases in cytosolic Ca2+ ([Ca2+]cyt), we identified Actin-Related Protein2 (Arp2) as a regulator of [Ca2+]cyt in response to salt stress. Plants lacking Arp2 or other proteins in the Arp2/3 complex exhibited enhanced salt-induced increases in [Ca2+]cyt, decreased mitochondria movement, and hypersensitivity to salt. In addition, mitochondria aggregated, the mitochondrial permeability transition pore opened, and mitochondrial membrane potential Ψm was impaired in the arp2 mutant, and these changes were associated with salt-induced cell death. When opening of the enhanced mitochondrial permeability transition pore was blocked or increases in [Ca2+]cyt were prevented, the salt-sensitive phenotype of the arp2 mutant was partially rescued. These results indicate that the Arp2/3 complex regulates mitochondrial-dependent Ca2+ signaling in response to salt stress.

Background Peroxisome proliferator activated receptor alpha (PPARα) ameliorates ethanol induced hepatic steatohepatitis. However, its role in alcoholic liver fibrosis has not been fully clarified. The aim of this study was to elucidate the effect and the molecular basis of PPARα in ethanol induced liver fibrosis in mice. Methods C57BL/6J mice were fed with 4% ethanol-containing Lieber-DeCarli liquid diet for eight weeks, and intraperitoneal injected with 5% carbon tetrachloride (CCl4) for the last four weeks to induce alcoholic liver fibrosis. PPARα agonist WY14643 was administered to mice during the last couple of weeks. The effects of PPARα induction on liver histology, activation of hepatic stellate cells (HSCs), as well as hepatic expression of inflammatory and fibrogenic factors were assessed. Results The ethanol plus CCl4 treated mice exhibited progressive liver injury including piecemeal necrosis of hepatocytes, severe inflammatory cells infiltration and bridging fibrosis. This was accompanied by down-regulated hepatic expression of PPARα and the protective cytokines adiponectin, heme oxygenase-1 and interleukin-10. Additionally, up-regulation of the proinflammatory cytokine tumor necrosis factor-alpha, as well as the profibrogenic genes osteopontin, transforming growth factor-beta 1, visfatin, phosphatidylinositol 3-kinase, matrix metalloproteinase-2 (MMP-2) and MMP-9 was observed. WY14643 treatment restored expression of cytokines altered by ethanol plus CCl4 treatment and concomitantly ameliorated the liver injury. Conclusions The present study provides evidence for the protective role of PPARα induction in ameliorating ethanol mediated fibrosis through mediation of inflammatory and fibrogenic factors.

Recent findings indicate that cannabinoid-altered vocal development involves elevated densities of dendritic spines in a subset of brain regions involved in zebra finch song learning and production suggesting that cannabinoid receptor activation may regulate cell structure. Here we report that activation of zebra finch CB 1 receptors (zfCB 1, delivered by a lentivector to CHO cells) produces dose-dependent biphasic effects on the mean length of filopodia expressed: Low agonist concentrations (3 nM WIN55212-2) increase lengths while higher concentrations reduce them. In contrast, treatment of zfCB 1-expressing cells with the antagonist/inverse agonist SR141716A causes increases in both mean filopodia length and number at 30 and 100 nM. These results demonstrate that CB 1 receptor activation can differentially influence filiopodia elongation depending on dose, and demonstrate that manipulation of cannabinoid receptor activity is capable of modulating cell morphology.

Background Fuzheng Huayu recipe (FZHY), a compound of Chinese herbal medicine, was reported to improve liver function and fibrosis in patients with hepatitis B virus infection. However, its effect on nutritional fibrosing steatohepatitis is unclear. We aimed to elucidate the role and molecular mechanism of FZHY on this disorder in mice. Methods C57BL/6 J mice were fed with methionine-choline deficient (MCD) diet for 8 weeks to induce fibrosing steatohepatitis. FZHY and/or heme oxygenase-1 (HO-1) chemical inducer (hemin) were administered to mice, respectively. The effect of FZHY was assessed by comparing the severity of hepatic injury, levels of hepatic lipid peroxides, activation of hepatic stellate cells (HSCs) and the expression of oxidative stress, inflammatory and fibrogenic related genes. Results Mice fed with MCD diet for 8 weeks showed severe hepatic injury including hepatic steatosis, necro-inflammation and fibrosis. Administration of FZHY or hemin significantly lowered serum levels of alanine aminotransferase, aspartate aminotransferase, reduced hepatic oxidative stress and ameliorated hepatic inflammation and fibrosis. An additive effect was observed in mice fed MCD supplemented with FZHY or/and hemin. These effects were associated with down-regulation of pro-oxidative stress gene cytochrome P450 2E1, up-regulation of anti-oxidative gene HO-1; suppression of pro-inflammation genes tumor necrosis factor alpha and interleukin-6; and inhibition of pro-fibrotic genes including α-smooth muscle actin, transforming growth factor beta 1, collagen type I (Col-1) and Col-3. Conclusions Our study demonstrated the protective role of FZHY in ameliorating nutritional fibrosing steatohepatitis. The effect was mediated through regulating key genes related to oxidative stress, inflammation and fibrogenesis.

Background Peroxisome proliferator activated receptor alpha (PPARα) regulates lipids metabolism and inhibits inflammatory response. However, the role of PPARα in alcoholic liver disease is largely unknown. We aim to elucidate the effect and the molecular basis of PPARα in ethanol induced hepatic injury in mice. Results C57BL/6J mice fed with 4% ethanol-containing Lieber-DeCarli liquid diet for 12 weeks exhibited hepatocyte steatosis, necrosis and inflammatory infiltration, accompanied with elevated serum alanine aminotransferase (ALT) and aspartic transaminase (AST) levels, decreased hepatic expression of PPARα, lipids oxidation promoting genes and anti-inflammatory factors, as well as enhanced hepatic expression of fatty acids synthesis promoting genes and pro-inflammatory cytokines. Induction of PPARα by PPARα agonist WY14643 treatment for 2 weeks ameliorated the severity of liver injury and restored expression of genes altered by ethanol treatment. However, administration of PPARα antagonist GW6471 for 2 weeks promoted the inflammatory response. Conclusions The present study provided the evidence for the protective role of PPARα in ameliorating ethanol induced liver injury through modulation of the genes related to lipid metabolism and inflammatory response.

Tight junctions (TJs) are the most apical component of the junctional complexes in mammalian epithelial cells and form selective paracellular barriers restricting the passage of solutes and ions across the epithelial sheets. Claudins, a TJ integral membrane protein family, play a critical role in regulating paracellular barrier permeability. In the in vitro cell culture system, transepithelial electrical resistance (TER) measurement and the flux of radioisotope or fluorescent labeled molecules with different sizes have been widely used to determine the TJ barrier function. In the in vivo system, the tracer molecule Sulfo-NHS-Biotin was initially used in Xenopus embryos system and subsequently was successfully applied to a number of animal tissues in situ and in different organisms under the experimental conditions to examine the functional integrity of TJs by several laboratories. In this chapter, we will describe the detailed procedures of applying biotin as a paracellular tracer molecule to different in vivo systems to assay TJ barrier function.

Claudin-7, a member of the claudin family, is highly expressed in distal nephrons of kidneys and has been reported to be involved in the regulation of paracellular Cl−permeability in cell cultures. To investigate the role of claudin-7 in vivo, we generated claudin-7 knockout mice (Cln7−/−) by the gene-targeting deletion method. Here we report that Cln7−/−mice were born viable, but died within 12 days after birth. Cln7−/−mice showed severe salt wasting, chronic dehydration, and growth retardation. We found that urine Na+, Cl−, and K+were significantly increased in Cln7−/−mice compared with that of Cln7+/+mice. Blood urea nitrogen and hematocrit were also significantly higher in Cln7−/−mice. The wrinkled skin was evident when Cln7−/−mice were ∼1 wk old, indicating that they suffered from chronic fluid loss. Transepidermal water loss measurements showed no difference between Cln7+/+and Cln7−/−skin, suggesting that there was no transepidermal water barrier defect in Cln7−/−mice. Claudin-7 deletion resulted in the dramatic increase of aldosterone synthase mRNA level as early as 2 days after birth. The significant increases of epithelial Na+channel α, Na+-Cl−cotransporter, and aquaporin 2 mRNA levels revealed a compensatory response to the loss of electrolytes and fluid in Cln7−/−mice. Na+-K+-ATPase α1expression level was also greatly increased in distal convoluted tubules and collecting ducts where claudin-7 is normally expressed. Our study demonstrates that claudin-7 is essential for NaCl homeostasis in distal nephrons, and the paracellular ion transport pathway plays indispensable roles in keeping ionic balance in kidneys.

18 pages, 9 figures, 35 references. Quan, Ruidang et al., The SOS (for Salt Overly Sensitive) pathway plays essential roles in conferring salt tolerance in Arabidopsis thaliana. Under salt stress, the calcium sensor SOS3 activates the kinase SOS2 that positively regulates SOS1, a plasma membrane sodium/ proton antiporter. We show that SOS3 acts primarily in roots under salt stress. By contrast, the SOS3 homolog SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCABP8)/CALCINEURIN B-LIKE10 functions mainly in the shoot response to salt toxicity. While root growth is reduced in sos3 mutants in the presence of NaCl, the salt sensitivity of scabp8 is more prominent in shoot tissues. SCABP8 is further shown to bind calcium, interact with SOS2 both in vitro and in vivo, recruit SOS2 to the plasma membrane, enhance SOS2 activity in a calcium-dependent manner, and activate SOS1 in yeast. In addition, sos3 scabp8 and sos2 scabp8 display a phenotype similar to sos2, which is more sensitive to salt than either sos3 or scabp8 alone. Overexpression of SCABP8 in sos3 partially rescues the sos3 salt-sensitive phenotype. However, overexpression of SOS3 fails to complement scabp8. These results suggest that SCABP8 and SOS3 are only partially redundant in their function, and each plays additional and unique roles in the plant salt stress response., This work was supported by National Basic Research Program of China Grant 2006CB100100 and National High Technology Research and Development Program of China 863 Grant 2003AA210100 to Y.G. and S.C. and by Spanish Ministry of Education and Science Grant BFU2006-06968 to J.M.P.

Background & Aims The cellular immunity has a profound impact on the status of hepatitis C virus (HCV) infection. However, the response of cellular immunity on the virological response in patients with antiviral treatment remains largely unclear. We aimed to clarify the response of peripheral T cells and monocytes in chronic hepatitis C patients with antiviral treatment. Methods Patients with chronic hepatitis C were treated either with interferon alpha-2b plus ribavirin (n = 37) or with pegylated interferon alpha-2a plus ribavirin (n = 33) for up to 24 weeks. Frequencies of peripheral regulatory T-cells (Tregs), programmed death-1 (PD-1) expressing CD4+ T-cells or CD8+ T-cells and toll-like receptor (TLR) 3 expressing CD14+ monocytes were evaluated by flow cytometry in patients at baseline, 12 and 24 weeks following treatment and in 20 healthy controls. Results Frequencies of Tregs, PD-1 and TLR3 expressing cells were higher in patients than those in control subjects (P

The use of celecoxib is associated with a significant decrease in breast cancer risk. However, the long-term use of high-dose celecoxib might be limited owing to cardiovascular side effects. In this study, we found that acetylbritannilactone (ABL), extract from a Chinese medicinal herb, could reduce celecoxib dose and potentiate the growth-inhibitory effect in breast cancer cells. ABL enhanced the apoptotic effect of celecoxib in COX-2-expressing cells, but had little effect in COX-2-negative cells. The apoptosis induced by the combination treatment disappeared when COX-2 was knocked down, whereas the lack of apoptotic effects in COX-2-negative cells was reversed after COX-2 transfection. However, the combination treatment induced a G(0)/G(1) phase arrest independent of whether or not the cells expressed COX-2. The G(0)/G(1) arrest was attributed to a decreased expression of cyclinD1, cyclinE, CDK2 and CDK6, especially the upregulation of p21. In addition, inhibition of Akt and p38 signaling pathways was required by the synergism, as the constitutively active Akt and p38 protected cells against apoptosis and cell cycle arrest induced by the combination treatment. In vivo, administration of celecoxib and ABL were more effective than the individual agents against xenograft tumor growth. Thus, our data suggested that the combinatorial approach of celecoxib and ABL might be helpful for breast cancer treatment.

Mutations in WNK4 have been linked to hypertension in PHAII. Paracellular ion transport has been reported to be involved in this disease process; however, the specific molecular target has not been identified. In this study, we found that TJ protein claudin-7 and WNK4 were partially co-localized in renal tubules of rat kidney and co-immunoprecipitated in kidney epithelial cells. The wild-type and PHAII-causing mutant, but not the kinase-dead mutant, phosphorylated claudin-7. We have identified ser206 in the COOH-terminus of claudin-7 as a putative phosphorylation site for WNK4. More importantly, disease-causing mutant enhanced claudin-7 phosphorylation and significantly increased paracellular permeability to Cl−.