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

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 CCl