Activation of nuclear-factor-E2-related factor 2 (Nrf2) cascade can alleviate dexamethasone (DEX)-induced oxidative injury and death of human osteoblasts. A recent study has shown that phosphoglycerate kinase 1 (PGK1) inhibition/depletion will lead to Kelch-like ECH-associated protein 1 (Keap1) methylglyoxal modification, thereby activating Nrf2 signaling cascade. Here, in OB-6 osteoblastic cells and primary human osteoblasts, PGK1 silencing, by targeted shRNA, induced Nrf2 signaling cascade activation, causing Nrf2 protein stabilization and nuclear translocation, as well as increased expression of ARE-dependent genes (HO1, NQO1, and GCLC). Functional studies demonstrated that PGK1 shRNA largely attenuated DEX-induced oxidative injury and following death of OB-6 cells and primary osteoblasts. Furthermore, PGK1 knockout, by the CRISPR/Cas9 method, similarly induced Nrf2 signaling activation and protected osteoblasts from DEX. Importantly, PGK1 depletion-induced osteoblast cytoprotection against DEX was almost abolished by Nrf2 shRNA. In addition, Keap1 shRNA mimicked and nullified PGK1 shRNA-induced anti-DEX osteoblast cytoprotection. At last we show that PGK1 expression is downregulated in human necrotic femoral head tissues of DEX-taking patients, correlating with HO1 depletion. Collectively, these results show that PGK1 depletion protects human osteoblasts from DEX via activation of Keap1-Nrf2 signaling cascade.

Heparan sulfate (HS) interacts with growth factors and has been implicated in regulating chondrogenesis. However, the effect of HS on TGF‐β‐mediated mesenchymal stem cell (MSC) chondrogenesis and molecular mechanisms remains unknown. In this study, we explored the effects of exogenous HS alone and in combination with TGF‐β3 on chondrogenic differentiation of human MSCs and possible signal mechanisms. The results indicated that HS alone had no obvious effects on chondrogenic differentiation of human MSCs and TGF‐β/Smad2/3 signal pathways. However, the combined TGF‐β3/HS treatment resulted in a significant increase in GAG synthesis, cartilage matrix protein secretion, and cartilage‐specific gene expression compared to cells treated with TGF‐β3 alone. Furthermore, HS inhibited type III TGF‐β receptors (TβRIII) expression and increased TGF‐β3‐mediated ratio of the type II (TβRII) to the type I (TβRI) TGF‐β receptors and phosphorylation levels of Smad2/3. The inhibitor of the TGF‐β/Smad signal, SB431542, not only completely inhibited HS‐stimulated TGF‐β3‐mediated Smad2/3 phosphorylation but also completely inhibited the effects of HS on TGF‐β3‐induced chondrogenic differentiation. These results demonstrate exogenous HS enhances TGF‐β3‐induced chondrogenic differentiation of human MSCs by activating TGF‐β/Smad2/3 signaling.