Tumor Necrosis Factor-α Attenuates the Osteogenic Differentiation Capacity of Periodontal Ligament Stem Cells by Activating PERK Signaling

Background: Human periodontal ligament stem cells (PDLSCs) display efficient osteogenic differentiation capacity but fail to rescue bone breakdown associated with periodontitis. Endoplasmic reticulum (ER) stress and the unfolded protein response have recently been linked to inflammation and osteogenic differentiation. Therefore, the role of the double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) pathway in the impaired osteogenic differentiation ability of PDLSCs treated with tumor necrosis factor (TNF)-alpha was investigated. Methods: PDLSCs were isolated and stimulated with osteogenic media containing 1, 10, or 20 ng/mL TNF-alpha. Assessment included: 1) expression of runt-related transcription factor 2 and osteocalcin; 2) mRNA expression and activity of alkaline phosphatase; and 3) formation of mineralization nodules. Furthermore, expression of PERK pathway-related factors: 1) glucose-regulated protein (GRP) 78; 2) PERK; 3) activating transcription factor (ATF) 4; and 4) CCAAT-enhancer-binding proteins (C/EBP) homologous protein were also measured. Osteogenic differentiation and inhibition of the PERK pathway were also examined in cells pretreated with an inhibitor of ER stress, 4-phenylbutyric acid (PBA), followed by TNF-alpha stimulation. Finally, PERK small interfering RNA was used to examine osteogenic differentiation attenuated by TNF-alpha. Results: Higher concentrations of TNF-alpha (10 and 20 ng/mL) impaired osteogenic differentiation of PDLSCs but activated the PERK pathway. Pretreatment of PDLSCs with lower concentrations of 4-PBA prevented the TNF-alpha-induced upregulation of GRP78, PERK, and ATF4 and recovered differentiation ability. Finally, PERK knockdown also restored osteogenic differentiation. Conclusion: TNF-alpha attenuates osteogenic differentiation ability of PDLSCs through activation of the PERK pathway.