Thymosin Beta 4 Influences Hedgehog Signaling by Interacting with Smo‐Gli2 in Hepatic Stellate Cells
Authors: Kim Jieun , Jung Youngmi 
Published: November 2023

Liver fibrosis, a major characteristic of chronic liver diseases, is regulated by a complex network of signaling pathways. During liver injury, hepatic stellate cells (HSCs) are transdifferentiated from quiescent HSCs into activated HSCs, which promote the deposition of extracellular matrix proteins, leading to liver fibrosis. Hedgehog (Hh) pathway is involved in the proliferation and activation of HSCs, contributing to the hepatic fibrosis. Thymosin beta 4 (Tβ4), the major actin‐sequestering polypeptide, has been reported to be involved in multiple biological processes including wound healing and fibrosis. However, the biological effects of this peptide in the liver are poorly understood. In the previous studies, we have showed that the endogenous level of Tβ4 is upregulated in fibrotic liver tissues of both humans and mice and it influences HSC activation. However, the detailed mechanism underlying how Tβ4 regulates the activation HSC remains unknown. Hence, we investigate whether and how Tβ4 interacts with Hh signaling pathway during HSC transdifferentiation.LX‐2 cells, fully activated HSC line originated from human liver, were transfected with Tβ4 siRNA or scramble (Con) siRNA, and examined for functions and activation of HSCs. Also, clustered regularly interspaced short palindromic repeats (CRISPR) RNA‐guided Cas9 nucleases was employed to knock out Tβ4 gene (Tβ4 CRISPR KO) in LX‐2 cells.Both of the proliferation and the migration of HSCs were significantly reduced in Tβ4 siRNA‐treated LX‐2 cells with the decreased expression of α‐smooth muscle actin (α‐SMA), an activation marker of HSC, compared with Con siRNA‐treated cells. Tβ4 suppression alleviated the level of both integrin‐linked kinase (ILK), a positive regulator of Smo, and phosphorylated GSK‐3β (pGSK‐3β), an inactive form of GSK‐3β which was known to degrade Gli2, followed by the decreased the expression of Hh effector, smoothened (Smo), and Hh‐target gene, glioblastoma 2 (Gli2), in both RNA and protein level. Double immunostaining and immunoprecipitation assay revealed that Tβ4 interacted with either Smo at cytoplasm or Gli2 at nucleus in LX‐2, implicating that Tβ4 might be involved in Hh signaling by interacting with Smo and Gli2. In addition, Tβ4 CRISPR KO induced the decreased expression of Smo, Gli2, ILK, and profibrotic genes, α‐SMA, n‐cadherin and transforming growth factor‐β, and the increased expression of the markers of quiescent HSCs, glial fibrillary acidic protein and peroxisome proliferator‐activated receptor‐γ, in LX‐2 cells, compared with non‐targeting CRISPR KO‐transfected cells. Oil Red O staining showed the cytoplasmic lipid droplets, the morphological hallmark of inactivated HSCs, in Tβ4 CRISPR KO‐transfected cells.Taken together, these findings demonstrate that Tβ4 influences the activation of Smo‐Gli2 by modulating the expression of ILK and pGSK‐3β, and is involved in regulating HSC activation and functions, suggesting Tβ4 as novel therapeutic target for developing treatment for chronic liver disease.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2014M2B2A9030333).