Nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is key in maintaining redox homeostasis and the pathogenesis of osteoarthritis (OA) involves oxidative distress. We thus investigated whether Nrf2/ARE signaling may control expression of key chondrogenic differentiation and hyaline cartilage maintenance factor SOX9.

In human C-28/I2 chondrocytes SOX9 expression was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap “n” collar homology-associated protein 1 (Keap1). Putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays to verify whether Nrf2 transcriptionally regulates SOX9. SOX9 promoter activity without and with Nrf2-inducer methysticin were analyzed. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Data were analyzed by one-way ANOVA, a Student's t-test, or Wilcoxon rank-sum test, according to the normal distribution. Statistical significance was set to p < 0.05.

While Keap1-specific RNAi increased SOX9 expression, Nrf2-specific RNAi significantly decreased it. Putative ARE sites (ARE₁, ARE₂) were identified in the SOX9 promoter region. ARE₂ mutagenesis significantly reduced SOX9 promoter activity, while truncation of ARE₁ did not. A functional ARE₂ site was thus essential for methysticin-mediated induction of SOX9 promoter activity. Knee cartilage of young Nrf2-knockout mice further revealed significantly fewer Sox9-positive chondrocytes as compared to old Nrf2-knockout animals, which further showed thinner cartilage and more severe cartilage erosion.

Our data suggest that SOX9 expression in articular cartilage is directly Nrf2-dependent and that pharmacological Nrf2 activation may hold potential to diminish age-dependent osteoarthritic changes in knee cartilage through improving protective SOX9 expression.

Background

Transcription factor nuclear factor E2p45-related factor 2 (Nrf2) is crucial for controlling the antioxidant response and maintaining cellular redox homeostasis. Binding of Nrf2 to antioxidant response elements (ARE) promotes the expression of anti-oxidative stress enzymes. In osteoblasts, Nrf2 directly interacts with Runx2, a strong transcriptional activator of osteoblast-specific genes. Sox9, a key regulator of chondrocyte differentiation is dominant over Runx2 in mesenchymal chondrogenic precursors. We therefore aimed to elucidate the role of Nrf2, and its regulation of Sox9, in chondrocytes.

Introduction: Osteomyelitis often causes functional impairment due to tissue destruction and the incidence of this condition appears to be increasing. Antimicrobial peptides (AP) are effectors of the innate defence system and play a key role in host protection at cellular surfaces. Human beta-defensins (HBD) represent a major subclass of antimicrobial peptides and act as a first line defence through their broad spectrum of potent antimicrobial activity (1). The aim of the present in vitro and in vivo investigations was to study the expression and regulation of HBD-2 and -3 in the case of bacterial bone infection and to analyze the effects of immunosuppressive drugs on bone-derived AP-expression.

Methods: Samples of healthy human bone, osteomyelitic bone and cultured osteoblasts (primary-, hFOB- and SAOS-2 cells) were assessed for the expression of HBD-2/-3 by RT-PCR, immunohistochemistry or ELISA. Regulation of HBD-2/-3 was studied after exposure to Staphylococcus aureus (SAS) or Pseudomonas aeruginosa (PAS), proinflammatory cytokines (IL-1, 10ng/ml) and immunosuppressive drugs (glucocorticoids, methotrexate) and was assayed by ELISA. An osteomyelitis mouse model was performed to demonstrate the regulation of the murine homologues of HBD-2/-3 by real time RT-PCR and immunohistochemistry.

Results: ELISA experiments demonstrated, that samples of infected bone produce higher levels of endogenous antibiotics such as HBD-2 when compared with samples of healthy bone. After exposure of osteoblasts to bacteria or proinflammatory cytokines a clear HBD-2/-3 induction was observed. Additional treatment with glucocorticoids or methotrexate prevented bacteria mediated HBD-2 induction in cultured osteoblasts. The osteomyelitis mouse model demonstrated transcriptional up-regulation of the murine HBD-homologues in bone after intra-osseous contamination of the tibia.

Discussion: Our study firstly demonstrate that osteoblasts are able to produce anti-inflammatory peptides such as HBD-2 in vitro and in an animal model of staphylococcal osteomyelitis. We provide evidence for a new role of osteoblasts during infection of bone tissues, namely, the ability to produce antimicrobial peptides and modulating immune responses in inflammatory bone diseases.

Immunosuppressive drugs such as glucocorticoids or methotrexate may increase the susceptibility to bone infection by decreasing AP-expression levels in case of microbial challenge. Novel approaches to management are required particularly in the era of multi-resistant bacterial strains. Current investigation will focus on the regulation of human β-Defensins in bone and may allow artificial amplification for prevention of bacterial bone infection in the future.