Proline-rich 15 depletion enhances Nrf2 cascade to promote osteoblast survival from oxidative injury

Reducing oxidative stress via enhancing Nrf2 cascade activation can efficiently protect osteoblasts. Here siRNA-based screening of nuclear proteins identified PRR15 (proline-rich 15) as a potential regulator of the Nrf2-ARE pathway in osteoblasts. In primary murine osteoblasts, silencing PRR15 using targeted shRNA or CRISPR/Cas9-mediated knockout (KO) enhanced hydrogen peroxide (H₂O₂)-induced activation of the Nrf2 cascade, reducing oxidative stress and cell death. PRR15 silencing or KO also counteracted H₂O₂-induced suppression of proliferation and downregulation of differentiation markers (Runx2 and Col1a1) in murine osteoblasts. Similarly, PRR15 silencing in primary human osteoblasts amplified Nrf2-ARE activation and mitigated H₂O₂-induced oxidative stress. In contrast, PRR15 overexpression inhibited Nrf2 activation, leading to increased apoptosis under oxidative stress in murine osteoblasts. Importantly, Nrf2 silencing by targeted shRNA reversed the protective effects of PRR15 silencing/KO against H₂O₂-induced cytotoxicity in murine osteoblasts. PRR15 interacts with BACH1 (BTB and CNC Homology 1) to negatively regulate Nrf2 signaling, as PRR15 overexpression or silencing had no effect on Nrf2-ARE pathway activation in BACH1-silenced murine osteoblasts. In vivo, osteoblast-specific PRR15 knockdown via adeno-associated virus (AAV) injection reduced oxidative stress and ameliorated trabecular bone loss in ovariectomized mice. Thus PRR15 silencing enhances Nrf2 cascade to promote the survival of osteoblasts exposed to oxidative stress.