甲状旁腺激素相关蛋白（PTHrP）通过涉及蛋白激酶C-ζ（PKC-ζ）的负反馈回路缓冲Wnt/β-连环蛋白活性，以维持关节软骨稳态并减轻骨关节炎。

PTHrP buffers Wnt/β-catenin activity through a PKC-ζ involved negative feedback loop to maintain articular cartilage homeostasis and attenuate osteoarthritis.

作者信息

Tong W X, Chen H J, Huang Z L, Zhao D, Hu J

机构信息

Department of Orthopaedics, The First Affiliated Hospital, Shantou University Medical College, Guangdong Province, China.

出版信息

J Orthop Translat. 2025 Jul 22;54:65-76. doi: 10.1016/j.jot.2025.03.012. eCollection 2025 Sep.

DOI:10.1016/j.jot.2025.03.012

PMID:40735514

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12304701/

Abstract

BACKGROUND

Osteoarthritis (OA) is the most common joint disease worldwide and a leading cause of disability. The Wnt/β-catenin cascade is essential in articular cartilage development and homeostasis. Interruption of β-catenin (either overexpression or inhibition) leads to cartilage degeneration. However, the mechanism for stabilizing Wnt/β-catenin remains unclear.

METHODS

We established the mouse destabilization of the medial meniscus (DMM) OA model and analyzed the clinical specimens to detect Wnt/β-catenin and PTHrP. The chondrocytes were isolated and treated with various cytokines including Wnt3a, Ihh, IL-1β, and PTHrP to reveal the molecular mechanism. Epigenetic and bioinformatic analyses were conducted to screen the key genes for the PTHrP regulation, and an Adeno-associated Virus (AAV) delivery system for PTHrP was established for OA gene therapy (prevention) application.

RESULTS

We confirmed the Wnt/β-catenin activation and PTHrP suppression in cartilage in post-traumatic OA. Wnt/β-catenin further upregulated PTHrP expression through binding to its promoter (P2), and induced mRNA (AT6) transcript expression. Unexpectedly, PTHrP repressed Wnt/β-catenin activity and formed a Wnt/β-catenin-PTHrP negative feedback loop in very primary chondrocytes to maintain cartilage homeostasis. However, this negative feedback loop vanished in dedifferentiated chondrocytes, hypertrophic chondrocytes, and IL-1β treated primary chondrocytes. In these chondrocytes under pathological conditions, we further found that miR-106b-5p was increased and directly targeted PTHrP mRNA to abolish the feedback loop. Using Bulk RNA-seq and KEGG analysis, we screened and confirmed that PKC-ζ was activated by PTHrP through phosphorylation at Thr410/403, and subsequently induced β-catenin phosphorylation, ubiquitination and degeneration. Finally, we disclosed that exogenous PTHrP attenuated OA progression.

CONCLUSION

This study reveals that PTHrP is a vital mediator in keeping Wnt/β-catenin homeostasis through a negative feedback loop similar to its role in balancing the Ihh pathway activity in the secondary ossification center and growth plate.

THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE

These findings highlight that PTHrP might be a therapeutic target for attenuating cartilage degeneration and OA process by the gene therapy approach.

摘要

背景

骨关节炎（OA）是全球最常见的关节疾病，也是导致残疾的主要原因。Wnt/β-连环蛋白信号通路在关节软骨发育和稳态维持中至关重要。β-连环蛋白的中断（过表达或抑制）会导致软骨退变。然而，稳定Wnt/β-连环蛋白的机制仍不清楚。

方法

我们建立了小鼠内侧半月板不稳定（DMM）OA模型，并分析临床标本以检测Wnt/β-连环蛋白和甲状旁腺激素相关蛋白（PTHrP）。分离软骨细胞并用包括Wnt3a、印度刺猬因子（Ihh）、白细胞介素-1β（IL-1β）和PTHrP在内的多种细胞因子进行处理，以揭示分子机制。进行表观遗传学和生物信息学分析以筛选PTHrP调控的关键基因，并建立用于OA基因治疗（预防）应用的PTHrP腺相关病毒（AAV）递送系统。

结果

我们证实了创伤后OA中软骨内Wnt/β-连环蛋白的激活和PTHrP的抑制。Wnt/β-连环蛋白通过与其启动子（P2）结合进一步上调PTHrP表达，并诱导信使核糖核酸（mRNA）（AT6）转录本表达。出乎意料的是，PTHrP抑制Wnt/β-连环蛋白活性，并在非常早期的软骨细胞中形成Wnt/β-连环蛋白-PTHrP负反馈环以维持软骨稳态。然而，这种负反馈环在去分化软骨细胞、肥大软骨细胞和IL-1β处理的原代软骨细胞中消失。在这些病理条件下的软骨细胞中，我们进一步发现微小核糖核酸-106b-5p（miR-106b-5p）增加，并直接靶向PTHrP mRNA以消除反馈环。使用批量RNA测序（Bulk RNA-seq）和京都基因与基因组百科全书（KEGG）分析，我们筛选并证实蛋白激酶C-ζ（PKC-ζ）被PTHrP通过苏氨酸410/403位点的磷酸化激活，随后诱导β-连环蛋白磷酸化、泛素化和退变。最后，我们发现外源性PTHrP可减轻OA进展。