Accelerated aging in articular cartilage by ZMPSTE24 deficiency leads to osteoarthritis with impaired metabolic signaling and epigenetic regulation

Osteoarthritis (OA) is a prevalent age-related degenerative joint disease characterized by articular cartilage degradation and secondary bone changes, leading to pain, swelling, and impaired mobility. Currently, there are no effective disease-modifying treatments. The pathogenesis of OA is complex and not fully understood, involving an imbalance between chondrocyte anabolism and catabolism, as well as the accumulation of senescent cells. Existing animal models, such as DMM and ACLT, primarily focus on mechanical injury rather than age-related degeneration. Therefore, there is a critical need for an aging-induced OA model to accelerate the development of effective therapies. ZMPSTE24 is a metalloprotease crucial for the maturation of lamin A, a protein maintaining nuclear integrity. Deficiency in ZMPSTE24 leads to Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic disorder characterized by accelerated aging. While HGPS patients exhibit various aging-related features, the connection between ZMPSTE24 deficiency and OA remains unclear. This study hypothesized that ZMPSTE24 deficiency in chondrocytes could accelerate the aging process, leading to OA development. The study aimed to investigate the role of ZMPSTE24 in cartilage homeostasis, chondrocyte senescence, and OA pathogenesis, aiming to establish a novel aging-induced OA model.

The existing literature supports a strong correlation between cellular senescence and OA development. Studies have shown that the accumulation of senescent cells in articular cartilage and synovium contributes significantly to OA progression. Targeting senescent cells has emerged as a promising therapeutic strategy. However, the specific molecular mechanisms linking cellular senescence to OA pathogenesis remain largely unclear. While several transgenic animal models of OA exist, focusing on specific signaling pathways like EGFR, PPARγ, and β-catenin, an accurate model mimicking age-related OA is lacking. The role of ZMPSTE24 and its impact on lamin A processing in OA development is a relatively unexplored area. Although ZMPSTE24 deficiency leads to HGPS, a disease marked by premature aging, the direct link between ZMPSTE24 and OA pathogenesis is not established.

This study employed a multi-faceted approach involving both in vitro and in vivo experiments. **Mouse models:** Zmpste24 knockout (*Zmpste24^-/-*) mice were generated and crossed with Prx1-Cre and Col2-CreERT2 mice to create conditional knockout models (*Prx1-Cre; Zmpste24^fl/fl* and *Col2-CreERT2; Zmpste24^fl/fl*) for targeted ZMPSTE24 deletion in mesenchymal stem cells and chondrocytes, respectively. Tamoxifen was used to induce Cre recombinase activity in the latter model. **In vitro studies:** Primary chondrocyte progenitor cells were isolated from neonatal mice and subjected to Zmpste24 knockdown using shRNA. The effects on chondrogenic differentiation, cell proliferation (CCK-8 assay), and senescence (SA-β-gal staining) were evaluated. Progerin overexpression was also performed in C3H10 cells to assess its impact on chondrocyte metabolism and senescence. **In vivo studies:** The OA phenotype in various knockout mouse models was assessed through radiographic and µ-CT analysis, histological examination (Safranin O staining), and immunohistochemistry for markers like Aggrecan, MMP13, and P16. OARSI scores were used for OA severity assessment. Mechanical sensitivity testing was used to evaluate joint pain. **Transcriptome sequencing:** RNA sequencing was performed on articular chondrocytes from *Zmpste24^-/-* mice and progerin-overexpressing C3H10 cells to identify differentially expressed genes. Bioinformatic analysis was used to determine affected pathways and functional enrichment. **Epigenetic analysis:** Immunohistochemistry and Western blotting were performed to assess the levels of H3K27me3, a histone modification associated with gene silencing, in the various mouse models and cell lines. The role of the Polycomb repressive complex 2 (PRC2) and EZH2 were investigated by Western blotting, immunofluorescence, and CHX treatment to assess protein stability and ubiquitination. **Statistical Analysis:** Unpaired, two-tailed Student's *t*-tests were used to compare differences between groups.

This study yielded several key findings: 1. **Decreased ZMPSTE24 expression with age:** The expression of ZMPSTE24 was significantly reduced in articular cartilage with age, accompanied by cartilage degeneration and decreased Aggrecan expression. 2. **ZMPSTE24 deficiency accelerates chondrocyte senescence and induces OA:** Both global and conditional knockout of Zmpste24 in mice resulted in severe OA phenotypes, including cartilage degeneration, subchondral bone sclerosis, osteophyte formation, and increased joint pain. In vitro studies demonstrated that ZMPSTE24 knockdown inhibited chondrogenic differentiation, reduced cell proliferation, and accelerated senescence in primary chondrocytes. 3. **Progerin accumulation impairs chondrocyte metabolism and promotes inflammation:** Overexpression of progerin in C3H10 cells downregulated genes involved in cartilage homeostasis and upregulated genes associated with inflammation and catabolism. Transcriptome sequencing revealed a significant dysregulation of the PI3K-Akt and Hippo signaling pathways. 4. **Upregulation of H3K27me3 in chondrocyte senescence:** The study observed an increase in H3K27me3 levels in senescent chondrocytes from Zmpste24 deficient mice, confirmed by both immunohistochemistry and Western blotting. This was associated with increased EZH2 protein levels, and the interaction between progerin and EZH2 was shown to enhance EZH2 stability and H3K27me3 modifications. Treatment with the EZH2 inhibitor GSK126 demonstrated partial alleviation of the OA phenotype in Zmpste24 deficient mice. 5. **PI3K-Akt pathway dysregulation:** RNA-sequencing analysis revealed dysregulation of the PI3K-Akt and Hippo signaling pathways in both Zmpste24 deficient and progerin overexpressing cells, suggesting a potential mechanistic link between ZMPSTE24, progerin accumulation, and impaired chondrocyte metabolism.

This study successfully established a link between ZMPSTE24 deficiency, progerin accumulation, impaired chondrocyte metabolism, and the development of age-related OA. The findings highlight the role of cellular senescence, particularly the epigenetic modification H3K27me3, in OA pathogenesis. The observed dysregulation of PI3K-Akt and Hippo pathways suggests potential therapeutic targets. The use of specific inhibitors targeting EZH2, a key component of the PRC2 complex responsible for H3K27me3 methylation, demonstrated a partial reversal of OA features in the Zmpste24 knockout mice, further supporting the significance of this epigenetic mechanism. This aging-induced OA model provides a valuable tool for future research on disease mechanisms and drug discovery.

This study provides compelling evidence for a novel link between ZMPSTE24 deficiency, progerin accumulation, and the development of age-related OA. The findings reveal the critical role of chondrocyte senescence and epigenetic modifications, specifically increased H3K27me3, in disease progression. This novel aging-induced OA model opens new avenues for investigating OA pathogenesis and identifying potential therapeutic targets, particularly those involved in the PI3K-Akt pathway and epigenetic regulation. Future studies could explore the clinical relevance of these findings by investigating ZMPSTE24 expression and epigenetic modifications in human OA samples.

The study primarily used mouse models, which may not fully recapitulate the complexity of human OA. While the study focused on the role of ZMPSTE24 and epigenetic modifications, other contributing factors to OA pathogenesis were not fully explored. The sample size in some experiments could be considered relatively small, especially in the in vivo studies. The effect of GSK126 on the OA features was only partially alleviated, suggesting that other mechanisms might be involved.