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Thymosin beta-4 regulates activation of hepatic stellate cells via hedgehog signaling.

ABSTRACT: The molecular mechanisms of thymosin beta-4 (TB4) involved in regulating hepatic stellate cell (HSC) functions remain unclear. Therefore, we hypothesize that TB4 influences HSC activation through hedgehog (Hh) pathway. HSC functions declined in a TB4 siRNA-treated LX-2. TB4 suppression down-regulated both integrin linked kinase (ILK), an activator of smoothened, and phosphorylated glycogen synthase kinase 3 beta (pGSK-3B), an inactive form of GSK-3B degrading glioblastoma 2 (GLI2), followed by the decreased expression of both smoothened and GLI2. A TB4 CRISPR also blocked the activation of primary HSCs, with decreased expression of smoothened, GLI2 and ILK compared with cells transfected with nontargeting control CRISPR. Double immunostaining and an immunoprecipitation assay revealed that TB4 interacted with either smoothened at the cytoplasm or GLI2 at the nucleus in LX-2. Smoothened suppression in primary HSCs using a Hh antagonist or adenovirus transduction decreased TB4 expression with the reduced activation of HSCs. Tb4-overexpressing transgenic mice treated with CCl₄ were susceptible to the development hepatic fibrosis with higher levels of ILK, pGSK3b, and Hh activity, as compared with wild-type mice. These findings demonstrate that TB4 regulates HSC activation by influencing the activity of Smoothened and GLI2, suggesting TB4 as a novel therapeutic target in liver disease.

SUBMITTER: Kim J

PROVIDER: S-EPMC5476572 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Thymosin beta-4 regulates activation of hepatic stellate cells via hedgehog signaling.

Kim Jieun J Hyun Jeongeun J Wang Sihyung S Lee Chanbin C Lee Jae-Wook JW Moon Eun-Yi EY Cha Heejae H Diehl Anna Mae AM Jung Youngmi Y

Scientific reports 20170619 1

The molecular mechanisms of thymosin beta-4 (TB4) involved in regulating hepatic stellate cell (HSC) functions remain unclear. Therefore, we hypothesize that TB4 influences HSC activation through hedgehog (Hh) pathway. HSC functions declined in a TB4 siRNA-treated LX-2. TB4 suppression down-regulated both integrin linked kinase (ILK), an activator of smoothened, and phosphorylated glycogen synthase kinase 3 beta (pGSK-3B), an inactive form of GSK-3B degrading glioblastoma 2 (GLI2), followed by t ...[more]

PMID: 28630423

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Project description:Background & aimsCirrhosis results from accumulation of myofibroblasts derived from quiescent hepatic stellate cells (Q-HSCs); it regresses when myofibroblastic HSCs are depleted. Hedgehog signaling promotes transdifferentiation of HSCs by activating Yes-associated protein 1 (YAP1 or YAP) and inducing aerobic glycolysis. However, increased aerobic glycolysis alone cannot meet the high metabolic demands of myofibroblastic HSCs. Determining the metabolic processes of these cells could lead to strategies to prevent progressive liver fibrosis, so we investigated whether glutaminolysis (conversion of glutamine to alpha-ketoglutarate) sustains energy metabolism and permits anabolism when Q-HSCs become myofibroblastic, and whether this is controlled by hedgehog signaling to YAP.MethodsPrimary HSCs were isolated from C57BL/6 or Smoflox/flox mice; we also performed studies with rat and human myofibroblastic HSCs. We measured changes of glutaminolytic genes during culture-induced primary HSC transdifferentiation. Glutaminolysis was disrupted in cells by glutamine deprivation or pathway inhibitors (bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide, CB-839, epigallocatechin gallate, and aminooxyacetic acid), and effects on mitochondrial respiration, cell growth and migration, and fibrogenesis were measured. Hedgehog signaling to YAP was disrupted in cells by adenovirus expression of Cre-recombinase or by small hairpin RNA knockdown of YAP. Hedgehog and YAP activity were inhibited by incubation of cells with cyclopamine or verteporfin, and effects on glutaminolysis were measured. Acute and chronic liver fibrosis were induced in mice by intraperitoneal injection of CCl4 or methionine choline-deficient diet. Some mice were then given injections of bis-2-[5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethyl sulfide to inhibit glutaminolysis, and myofibroblast accumulation was measured. We also performed messenger RNA and immunohistochemical analyses of percutaneous liver biopsies from healthy human and 4 patients with no fibrosis, 6 patients with mild fibrosis, and 3 patients with severe fibrosis.ResultsExpression of genes that regulate glutaminolysis increased during transdifferentiation of primary Q-HSCs into myofibroblastic HSCs, and inhibition of glutaminolysis disrupted transdifferentiation. Blocking glutaminolysis in myofibroblastic HSCs suppressed mitochondrial respiration, cell growth and migration, and fibrogenesis; replenishing glutaminolysis metabolites to these cells restored these activities. Knockout of the hedgehog signaling intermediate smoothened or knockdown of YAP inhibited expression of glutaminase, the rate-limiting enzyme in glutaminolysis. Hedgehog and YAP inhibitors blocked glutaminolysis and suppressed myofibroblastic activities in HSCs. In livers of patients and of mice with acute or chronic fibrosis, glutaminolysis was induced in myofibroblastic HSCs. In mice with liver fibrosis, inhibition of glutaminase blocked accumulation of myofibroblasts and fibrosis progression.ConclusionsGlutaminolysis controls accumulation of myofibroblast HSCs in mice and might be a therapeutic target for cirrhosis.

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Thymosin beta-4 regulates activation of hepatic stellate cells via hedgehog signaling.

Publications

Thymosin beta-4 regulates activation of hepatic stellate cells via hedgehog signaling.

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