The Mechanism of Up-Regulated P4ha1 Increasing Collagen Fiber Diameter and Extracellular Matrix Stiffness in Myofibroblasts
DING Jingru, WU Meizhen, TAN Qiong, YUE Wenhui, LI Liying, YANG Le*
Liver disease seriously endangers human health in modern society. P4ha1 (prolyl 4-hydroxylase a1) has been found to be involved in cardiac fibrosis, but the effects and mechanism of P4ha1 on liver fibrosis are still unclear. MFs (myofibroblasts) play an important role in liver fibrosis and promote the progression of liver fibrosis by secreting a large amount of extracellular matrix components such as collagen. The aim of this study is to explore the regulatory mechanism of P4ha1 on collagen fiber diameter and extracellular matrix stiffness in MFs. The cell localization of P4ha1 in fibrotic liver was analyzed using public single cell sequencing databases. In vitro primary mouse BMSCs (bone marrow mesenchymal stromal cells) were treated with TGFβ1 (transforming growth factor beta 1) to induce their differentiation into MFs. The expression and cellular localization of P4ha1 in MFs were determined by RT-qPCR and immunofluorescent staining. With the treatment of P4ha1 inhibitor DiethylpythiDC in MFs, this study analyzed and quantified the content of hydroxyproline (per mol type I collagen), representing the enzymatic activity of P4ha1, observed collagen morphology under scanning electron microscope and measured the average diameter of collagen fibers. In addition, the stiffness of extracellular matrix in MFs was measured by atomic force microscopy after the inhibition of P4ha1. The results showed that P4ha1 was mainly localized in MFs of fibrotic liver. In vitro, BMSCs were treated with TGFβ1 to induce their differentiation into MFs. P4ha1 expression was up-regulated during MFs activation, and P4ha1 was localized in the cytoplasmic matrix region within MFs. With the treatment of DiethylpythiDC in MFs, the level of hydroxyproline decreased, collagen fiber diameter reduced, and extracellular matrix stiffness decreased. These results demonstrate that P4ha1 plays a key role in modulating collagen fiber diameter and stiffness via the hydroxylation of proline, thus promotes the progression of liver fibrosis.
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