title: Tumor necrosis factor alpha and interleukin-1beta 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 (TNFalpha) and interleukin-1beta (IL-1beta) modulate the osteocyte response to mechanical loading.
  
  
  METHODS
  MLO-Y4 osteocytes were incubated with TNFalpha (0.5-30 ng/ml) or IL-1beta (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). TNFalpha and IL-1beta inhibited these responses. Calcium inhibitors decreased pulsatile fluid flow-induced NO production. TNFalpha and IL-1beta affected cytoskeletal stiffness, likely because 24 hours of incubation with TNFalpha and IL-1beta decreased the amount of F-actin. Incubation with IL-1beta for 24 hours stimulated osteocyte apoptosis.
  
  
  CONCLUSIONS
  Our results suggest that TNFalpha and IL-1beta inhibit mechanical loading-induced NO production by osteocytes via abrogation of pulsatile fluid flow-stimulated [Ca(2+)](i), and that IL-1beta 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.
authors.affiliation: ACTA-Universiteit van Amsterdam and Vrije Universiteit, Research Institute MOVE, Amsterdam, The Netherlands.
authors.forename: A D
authors.initials: AD
authors.lastname: Bakker
authors.forename: V C Da
authors.initials: VC
authors.lastname: Silva
authors.forename: R
authors.initials: R
authors.lastname: Krishnan
authors.forename: R G
authors.initials: RG
authors.lastname: Bacabac
authors.forename: M E
authors.initials: ME
authors.lastname: Blaauboer
authors.forename: Y-C
authors.initials: YC
authors.lastname: Lin
authors.forename: R A C
authors.initials: RA
authors.lastname: Marcantonio
authors.forename: J A
authors.initials: JA
authors.lastname: Cirelli
authors.forename: J
authors.initials: J
authors.lastname: Klein-Nulend
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001862:Bone Resorption / Q000378:metabolism; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D004533:Egtazic Acid / Q000494:pharmacology; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D053583:Interleukin-1beta / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D023421:Models, Animal; D009569:Nitric Oxide / Q000378:metabolism*; D010011:Osteocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D013314:Stress, Mechanical; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000199:Actins; D002121:Calcium Channel Blockers; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D004533:Egtazic Acid; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D002118:Calcium