Distinct Epigenetic Responses of Zone-Specific Meniscus Cells to Pharmacologic Modulation of Rho/ROCK Signaling

Introduction: : The meniscus is essential for load distribution, shock absorption, and joint stability in the knee. Due to its critical biomechanical functions, meniscal tears and degeneration are highly prevalent and, if left untreated, can accelerate osteoarthritis progression and joint dysfunction [1]. These injuries often disrupt the extracellular matrix (ECM), altering the mechanical environment and basal cellular contractility of resident cells, ultimately influencing their phenotype and regenerative potential [2]. However, the mechanisms by which these mechanical changes impact epigenetic regulation remain poorly understood. Notably, the inner (avascular) and outer (vascularized) zones of the meniscus exhibit distinct cellular and ECM properties, yet little is known about how zone-specific cells epigenetically respond to injury-induced mechanical alterations in the meniscus. Thus, in this study, we investigated how pharmacologic inhibition of the Rho/ROCK signaling pathway, a key regulator of cellular contractility, affects histone organization and modification in region-specific meniscus cells, to model these changes in vitro and to inform future zone-specific therapeutic strategies.

Materials and

Methods: : Outer and inner cells were isolated from the same bovine meniscus. P0 cells were cultured on 8-well chambered coverglass slides for 24 hours. Lysophosphatidic acid (LPA, ROCK signaling pathway agonist) and Y-27632 (ROCK inhibitor) were used at several time points and concentrations. One group was treated with a higher concentration (LPA: 30uM, Y-27632: 10uM) and shorter duration (1 hour). The other group featured a lower concentration (LPA: 5uM, Y-27632: 0.5uM) but longer duration (3 hours). Super-resolution STORM imaging (ONI) of histone-H2B (H2B) was performed to assess nano-scale chromatin structure [3]. Immunofluorescence (IF) imaging of acetylation of H3K27 (H3K27ac, epigenetic activation and chromatin decondensation) was carried out to investigate alterations in histone modification status.

Results, Conclusions, and Discussions:: STORM imaging of untreated meniscus cells revealed that inner zone cells exhibited higher chromatin condensation compared to outer zone cells (Fig. 1a, b). Upon treatment with LPA, chromatin condensation decreased in both inner and outer cells under high-concentration with short-duration conditions (LPA 1h-H). Interestingly, under low-concentration with the long-duration LPA treatment (LPA 3h-L), a significant reduction in chromatin condensation was observed only in outer cells (Fig. 2a, b). In contrast, Y-27632 treatment induced a more pronounced effect in only inner cells, particularly with low-dose with the long-duration (Y27 3h-L) exposure, resulting in marked chromatin compaction (Fig. 2c, d). To further investigate the epigenetic consequences of altered cellular contractility, we performed immunofluorescence staining for H3K27ac, a histone mark associated with active transcription and chromatin decondensation. Both LPA and Y-27632 treatments led to increased H3K27ac levels across cell types. However, in inner zone cells, this increase was attenuated under the low-dose with long-duration condition, which is similar to the chromatin condensation patterns observed by STORM (Fig. 3a, b). Collectively, these results demonstrate that cytoskeleton-mediated regulation of chromatin organization is zone-specific in the meniscus.
In this study, we demonstrated that meniscus cells exhibit distinct, zone-specific epigenetic responses to pharmacologic modulation of the Rho/ROCK signaling pathway. Outer zone cells were more responsive to increased contractility induced by LPA, whereas inner zone cells showed greater sensitivity to contractility inhibition via Y-27632. These differential responses likely reflect intrinsic differences in baseline cellular contractility, mediated by the local extracellular matrix (ECM) environment. The outer meniscus, characterized by fibrous, vascularized, and stiffer ECM, may promote higher baseline contractile activity, while the inner, cartilaginous and avascular region is associated with a more compliant matrix and lower intrinsic contractility [4]. These biomechanical distinctions in meniscus may influence chromatin organization and histone modification patterns in a zone-dependent manner. Taken together, our findings suggest the need to consider regional cellular heterogeneity in the development of meniscus repair strategies. Future studies integrating transcriptomic and functional assays will further clarify how mechanical signaling and epigenetic regulation intersect to control meniscus cell phenotype and regenerative capacity.

Acknowledgements and/or References (Optional):: [1] Makris+ 2011, Biomaterials; [2] Humphrey+ 2014, Nat Rev Mol Cell Biol.; [3] Heo+ 2022, Nat. Biomed. Eng.; [4] Lee+ 2025, Bioact. Mater.