Inhibitory effects of microRNA 19b in hepatic stellate cell-mediated fibrogenesis

Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGFβ). microRNAs (miRs), small non-coding RNAs modulating mRNA and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGFβ signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Since miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGFβ signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGFβ signaling components demonstrated by decreased TGFβ receptor II (TGFβRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3’UTR of TGFβRII was validated by luciferase reporter assay. Inhibition of TGFβ signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGFβ-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased αSMA expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared to normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared to normal human livers

MiR-155 has a protective role in the development of non-alcoholic hepatosteatosis in mice

Hepatic steatosis is a global epidemic that is thought to contribute to the pathogenesis of type 2 diabetes. MicroRNAs (miRs) are regulators that can functionally integrate a range of metabolic and inflammatory pathways in liver. We aimed to investigate the functional role of miR-155 in hepatic steatosis. Male C57BL/6 wild-type (WT) and miR-155−/− mice were fed either normal chow or high fat diet (HFD) for 6 months then lipid levels, metabolic and inflammatory parameters were assessed in livers and serum of the mice. Mice lacking endogenous miR-155 that were fed HFD for 6 months developed increased hepatic steatosis compared to WT controls. This was associated with increased liver weight and serum VLDL/LDL cholesterol and alanine transaminase (ALT) levels, as well as increased hepatic expression of genes involved in glucose regulation (Pck1, Cebpa), fatty acid uptake (Cd36) and lipid metabolism (Fasn, Fabp4, Lpl, Abcd2, Pla2g7). Using miRNA target prediction algorithms and the microarray transcriptomic profile of miR-155−/− livers, we identified and validated that Nr1h3 (LXRα) as a direct miR-155 target gene that is potentially responsible for the liver phenotype of miR-155−/− mice. Together these data indicate that miR-155 plays a pivotal role regulating lipid metabolism in liver and that its deregulation may lead to hepatic steatosis in patients with diabetes

Neonatal Androgenization Exacerbates Alcohol-Induced Liver Injury in Adult Rats, an Effect Abrogated by Estrogen

Alcoholic liver disease (ALD) affects millions of people worldwide and is a major cause of morbidity and mortality. However, fewer than 10% of heavy drinkers progress to later stages of injury, suggesting other factors in ALD development, including environmental exposures and genetics. Females display greater susceptibility to the early damaging effects of ethanol. Estrogen (E2) and ethanol metabolizing enzymes (cytochrome P450, CYP450) are implicated in sex differences of ALD. Sex steroid hormones are developmentally regulated by the hypothalamic-pituitary-gonadal (HPG) axis, which controls sex-specific cycling of gonadal steroid production and expression of hepatic enzymes. The aim of this study was to determine if early postnatal inhibition of adult cyclic E2 alters ethanol metabolizing enzyme expression contributing to the development of ALD in adulthood. An androgenized rat model was used to inhibit cyclic E2 production. Control females (Ctrl), androgenized females (Andro) and Andro females with E2 implants were administered either an ethanol or isocalorically-matched control Lieber-DeCarli diet for four weeks and liver injury and CYP450 expression assessed. Androgenization exacerbated the deleterious effects of ethanol demonstrated by increased steatosis, lipid peroxidation, profibrotic gene expression and decreased antioxidant defenses compared to Ctrl. Additionally, CYP2E1 expression was down-regulated in Andro animals on both diets. No change was observed in CYP1A2 protein expression. Further, continuous exogenous administration of E2 to Andro in adulthood attenuated these effects, suggesting that E2 has protective effects in the androgenized animal. Therefore, early postnatal inhibition of cyclic E2 modulates development and progression of ALD in adulthood

microRNAs: Fad or future of liver disease

microRNAs (miRs) are small non-coding RNAs that regulate both mRNA and protein expression of target genes, which results in alterations in mRNA stability or translation inhibition. miRs influence at least one third of all human transcripts and are known regulators of various important cellular growth and differentiation factors. miRs have recently emerged as key regulatory molecules in chronic liver disease. This review details recent contributions to the field of miRs that influence liver development and the broad spectrum of disease, from non-alcoholic fatty liver disease to fibrosis/cirrhosis, with particular emphasis on hepatic stellate cells and potential use of miRs as therapeutic tools

Nonmuscle myosin II regulates migration but not contraction in rat hepatic stellate cells

AIM: To identify and characterize the function of nonmuscle myosin II (NMM II) isoforms in primary rat hepatic stellate cells (HSCs)

Daily genetic profiling indicates JAK/STAT signaling promotes early hepatic stellate cell transdifferentiation

AIM: To identify signaling pathways and genes that initiate and commit hepatic stellate cells (HSCs) to transdifferentiation

Effects of ethanol and androgenization on CYP450 expression.

<p>A. Quantitative Real-Time PCR analysis of CYP2E1 mRNA expression in control (Ctrl), androgenized (Andro) and Andro + estrogen (Andro + E2) rats maintained on control Lieber-DeCarli (C-LDC) or ethanol-LDC (E-LDC) diet. B. Representative CYP2E1 protein expression was determined by Western blot analysis (upper panel) and quantified by optical integrated volume (lower panel). Data are presented as mean values ± SEM. Standard square root transformation was performed prior to statistical analysis. *P<0.05. C. Quantitative Real-Time PCR analysis of CYP1A2 mRNA expression compared between groups described in A. Data are presented as mean values ± SEM. *P<0.05. D. Representative CYP1A2 protein expression was determined by Western blot analysis (upper panel) and quantified by optical integrated volume (lower panel). RNA and protein expression were normalized to GAPDH.</p

Effects of ethanol and androgenization on antioxidant defense activity.

<p>A. Hepatic superoxide dismutase (SOD) activity determined by assay kit for control (Ctrl), androgenized (Andro) or androgenized + E2 (Andro + E2) animals maintained on control Lieber-DeCarli (C-LDC) or ethanol-LDC (E-LDC) diet. B. Hepatic catalase activity determined by assay kit for Ctrl, Andro, Andro + E2 rats maintained on C-LDC or E-LDC diets. Activity levels were normalized to total protein. Data are presented as mean values ± SEM. *P<0.05.</p

Effects of ethanol and androgenization on oxidative stress.

<p>A. Representative images of 4-HNE stained formalin-fixed paraffin-embedded liver tissue from rats maintained on control Lieber-DeCarli (C-LDC, top panels) or ethanol-LDC (E-LDC, bottom panels) diet. Panels A and D, control females (Ctrl); B and E, androgenized females (Andro); panels C and F, Andro + estrogen (Andro + E2). PT; portal triad. Caramel brown staining indicating 4-HNE positive cells. B. Hepatic lipid peroxidation was assessed by Thiobarbituric Acid Reactive Substances (TBARS) assay. Liver tissue was homogenized and malondialdehyde (MDA) formation measured. TBARS were normalized to total protein. Data are presented as mean values ± SEM. *P<0.05.</p

The interface of protein structure, protein biophysics, and molecular evolution

The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction