YAP1 Knockdown Reduces IL-1β-Induced Human Chondrocyte Inflammation and Promotes Human MSC Chondrogenesis

Background: Yes-associated protein 1 (YAP1), a key effector of the Hippo signaling pathway and mechanosensitive transcriptional coactivator, plays a complex role in osteoarthritis (OA) and cartilage regeneration. While YAP1 is essential for tissue homeostasis, its dysregulation has been implicated in both inflammatory and degenerative joint pathologies. However, its precise function remains ambiguous. Methods: We silenced YAP1 with small interfering RNA (siYAP1) in two human-cell-based models relevant to OA pathogenesis and cartilage repair: (1) IL-1β (10 ng/mL)-stimulated articular chondrocytes in monolayer and pellet cultures, and (2) TGF-β1 (10 ng/mL)-induced chondrogenesis in MSC pellet cultures. Outcome measures comprised YAP1 nuclear localization; inflammatory/catabolic markers in chondrocytes (IL6, IL8, ADAMTS5, MMP13); and, in MSC pellets, chondrogenic or hypertrophic markers (COL2A1, ACAN, RUNX2, MMP13, COL10A1) together with glycosaminoglycan (GAG) deposition. Statistical significance was assessed using an ANOVA or Friedman test with post hoc correction (Tukey or Dunn's test, respectively); p < 0.05 was considered significant. Results: In human chondrocytes, siYAP1 reduced IL-1β-induced nuclear YAP1 localization and suppressed pro-inflammatory mediators IL6 and IL8, indicating an anti-inflammatory effect. YAP1 silencing also downregulated ADAMTS5 expression in 2D monolayers but not in 3D pellet cultures, suggesting reduced regulatory influence in the three-dimensional environment. Notably, MMP13 expression was paradoxically increased following YAP1 knockdown, underscoring the complexity of YAP1's role in catabolic regulation. In MSC chondrogenesis, siYAP1 enhanced TGF-β1-induced chondrogenesis by increasing COL2A1 and ACAN expression and promoting GAG deposition on day 21. Additionally, it reduced hypertrophic markers RUNX2 and MMP13 on day 7, though COL10A1 remained elevated compared to negative siRNA, indicating only partial suppression of hypertrophic differentiation. Nuclear YAP1 levels were increased by day 21 despite reduced mRNA, suggesting post-transcriptional regulation or enhanced nuclear translocation. Conclusions: These findings demonstrate that YAP1 knockdown exerts context-specific anti-inflammatory and pro-chondrogenic effects while partially mitigating hypertrophy. However, divergent outcomes, namely elevated MMP13 in chondrocytes and upregulated COL10A1 in MSCs, indicate that YAP1 silencing does not uniformly suppress inflammation or hypertrophy. YAP1 represents a potential therapeutic target for OA, but its modulation requires careful consideration of cellular context, siRNA delivery method, and timing to optimize outcomes for cartilage repair and joint preservation.