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

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.