Tumor Necrosis Factor alpha and Interleukin-1 beta Modulate Calcium and Nitric Oxide Signaling in Mechanically Stimulated Osteocytes

Abstract

Objective. Inflammatory diseases often coincide with reduced bone mass. Mechanoresponsive osteocytes regulate bone mass by maintaining the balance between bone formation and resorption. Despite its biologic significance, the effect of inflammation on osteocyte mechanoresponsiveness is not understood. To fill this gap, we investigated whether the inflammatory cytokines tumor necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) modulate the osteocyte response to mechanical loading.Methods. MLO-Y4 osteocytes were incubated with TNF alpha (0.5-30 ng/ml) or IL-1 beta (0.1-10 ng/ml) for 30 minutes or 24 hours, or with calcium inhibitors for 30 minutes. Cells were subjected to mechanical loading by pulsatile fluid flow (mean +/- amplitude 0.7 +/- 0.3 Pa, 5 Hz), and the response was quantified by measuring nitric oxide (NO) production using Griess reagent and by measuring intracellular calcium concentration ([Ca(2+)](i)) using Fluo-4/AM. Focal adhesions and filamentous actin (F-actin) were visualized by immunostaining, and apoptosis was quantified by measuring caspase 3/7 activity. Cell-generated tractions were quantified using traction force microscopy, and cytoskeletal stiffness was quantified using optical magnetic twisting cytometry.Results. Pulsatile fluid flow increased [Ca(2+)](i) within seconds (in 13% of cells) and NO production within 5 minutes (4.7-fold). TNF alpha and IL-1 beta inhibited these responses. Calcium inhibitors decreased pulsatile fluid flow-induced NO production. TNF alpha and IL-1 beta affected cytoskeletal stiffness, likely because 24 hours of incubation with TNF alpha and IL-1 beta decreased the amount of F-actin. Incubation with IL-1 beta for 24 hours stimulated osteocyte apoptosis.Conclusion. Our results suggest that TNF alpha and IL-1 beta inhibit mechanical loading-induced NO production by osteocytes via abrogation of pulsatile fluid flow-stimulated [Ca(2+)](i), and that IL-1 beta stimulates osteocyte apoptosis. Since both NO and osteocyte apoptosis affect osteoclasts, these findings provide a mechanism by which inflammatory cytokines might contribute to bone loss and consequently affect bone mass in rheumatoid arthritis.