HPF1 通过 PARP1 介导的 HuR 多聚 ADP 核糖基化调控肌腱干/祖细胞衰老和肌腱修复。

HPF1 regulates tendon stem/progenitor cell senescence and tendon repair via PARP1-mediated poly-ADP ribosylation of HuR.

机构信息

Department of Orthopaedics, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070, China.

Senior Department of Orthopedics, The Fourth Medical Center of PLA General Hospital, No. 51 Fucheng Road, Beijing, 10048, China.

出版信息

Genes Genomics. 2024 Jan;46(1):27-36. doi: 10.1007/s13258-023-01447-w. Epub 2023 Sep 15.

DOI:10.1007/s13258-023-01447-w

PMID:37713069

Abstract

BACKGROUND

Tendon stem/progenitor cells (TSPCs) play a vital role in tendon repair, regeneration and homeostasis. However, the specific mechanism of TSPCs aging is still unclear.

OBJECTIVE

This study aims to explore the role and molecular mechanism of HPF1 in the aging of TSPCs.

METHODS

Young and aged TSPCs (Y-TSPCs and A-TSPCs) were acquired from 3 to 4 and 24-26-month-old Sprague-Dawley male rats, TSPCs (Y-TSPCs and A-TSPCs) were subjected to senescence-associated β-galactosidase (SA-β-Gal))staining and telomerase activity detection, p16, p21, Scx, Tnmd, Col1, Col3HPF1 and PAPR1 expression levels were detected by Western blot or Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), Reciprocal co-immunoprecipitation (co-IP) was used to explore the interaction between HPF1 and PARP1. Ribonucleoprotein immunoprecipitation (RNP-IP) was used to analyze the binding of HuR to the senescence marker gene mRNAs, IP was used to perform HPF1 to the PARylation of HuR, and the half-life of p16 and p21 were detected. Finally, we established an in vivo model, and the tendon tissue was used to perform hematoxylin and eosin (HE) and masson's trichrome staining, as well as the immunohistochemical analysis of Col I and TNMD.

RESULTS

Compared with Y-TSPCs, A-TSPCs had significantly enhanced cell senescence and significantly reduced tendon differentiation ability, and significantly increased the expression of HPF1 and PARP1. In addition, HPF1 and PARP1 interacted and coordinated the senescence and differentiation of TSPCs, HPF1 could also regulate the expression of p21 and p21, the interaction of p16 or p21 with HuR, and the poly-ADP ribosylation of PARP1 to HuR. HPF1 overexpression and siHuR co-transfection significantly reduced the half-life of p16 and p21, and HPF1 and PARP1 regulated the mRNA levels of p16 and p21 through HuR. Finally, in vivo experiments have shown that HPF1 or PARP1 overexpression could both inhibit the ability of tendon differentiation and promote cell senescence.

CONCLUSIONS

HPF1 promoted the senescence of TSPCs and inhibits the tendon differentiation of TSPCs through PARP1-mediated poly-ADP ribosylation of HuR.

摘要

背景

肌腱干/祖细胞（TSPCs）在肌腱修复、再生和稳态中起着至关重要的作用。然而，TSPCs 衰老的具体机制尚不清楚。

目的

本研究旨在探讨 HPF1 在 TSPCs 衰老中的作用及分子机制。

方法

从小鼠 3-4 月龄（青年 TSPCs，Y-TSPCs）和 24-26 月龄（老年 TSPCs，A-TSPCs）中分离培养 Y-TSPCs 和 A-TSPCs，采用衰老相关 β-半乳糖苷酶（SA-β-Gal）染色和端粒酶活性检测，Western blot 或反转录定量聚合酶链反应（RT-qPCR）检测 p16、p21、Scx、Tnmd、Col1、Col3、HPF1 和 PAPR1 表达水平，采用免疫共沉淀（co-IP）探讨 HPF1 与 PARP1 的相互作用，核糖核蛋白免疫沉淀（RNP-IP）分析 HuR 与衰老标志物基因 mRNA 的结合，免疫沉淀（IP）检测 HPF1 对 HuR 的 PAR 化修饰，以及 p16 和 p21 的半衰期。最后，构建体内模型，对肌腱组织进行苏木精-伊红（HE）和 Masson 三色染色以及 Col I 和 TNMD 的免疫组化分析。

结果

与 Y-TSPCs 相比，A-TSPCs 细胞衰老明显增强，肌腱分化能力明显降低，HPF1 和 PARP1 表达明显升高。此外，HPF1 与 PARP1 相互作用并协同调控 TSPCs 的衰老和分化，HPF1 还可调节 p21 和 p21 的表达、p16 或 p21 与 HuR 的相互作用、PARP1 对 HuR 的多聚 ADP 核糖基化修饰。HPF1 过表达和 siHuR 共转染可显著降低 p16 和 p21 的半衰期，HPF1 和 PARP1 通过 HuR 调控 p16 和 p21 的 mRNA 水平。最后，体内实验表明，HPF1 或 PARP1 过表达均可抑制肌腱分化能力并促进细胞衰老。