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

Jieun Kim,Eun-Yi Moon,Chanbin Lee,Youngmi Jung,Jae-Wook Lee,Heejae Cha,Sihyung Wang,Anna Mae Diehl,Jeongeun Hyun

doi:10.1038/s41598-017-03782-x

Jieun Kim, Eun-Yi Moon + Show 7 more

Open Access

https://doi.org/10.1038/s41598-017-03782-x

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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.

Highlights

Chronic liver disease is associated with substantial mortality and morbidity worldwide[1]
In the Thymosin beta-4 (TB4)-suppressed LX-2 cells, the protein levels of integrin linked kinase (ILK) and phosphorylated GSK3B (pGSK3B) were down-regulated after 24 h and after 24 and 36 h, respectively (Fig. 4b and c). These results demonstrated that knockdown of TB4 resulted in the reduced expression of ILK and pGSK3B before down-regulating SMO and glioblastoma 2 (GLI2) (Fig. 2), suggesting that TB4 was involved in SMO-GLI2 activation by regulating phosphorylation of glycogen synthase kinase 3 beta (GSK3B)-mediated by ILK during hepatic stellate cell (HSC) activation
We demonstrated that activated HSCs expressed TB4 in chronically damaged livers and that endogenous expression of TB4 was related with the activation of HSCs25

Summary

Introduction

Chronic liver disease is associated with substantial mortality and morbidity worldwide[1]. Hh signaling is initiated by the binding of Hh ligands, such as sonic Hh (SHH), indian Hh, and desert Hh, to the Hh receptor patched, activating smoothened (SMO), an effector of Hh signaling This activated SMO in turn promotes the production of the transcriptionally active forms of the glioblastoma (GLI) family (GLI1, GLI2, and GLI3)[9, 10]. These activated GLIs accumulate in the nucleus and regulate the expression of Hh-target genes[9, 11]. SMO is involved in production of GLIs-A, but the association of ILK-GSK3B with SMO in activating Glis is poorly understood. It demonstrated that TB4 stabilized ILK in cardiomyocyte and colon cancer cells and that overexpression of TB4 inactivated GSK3B by hyper-phosphorylating GSK3B31–33

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