title: Expression of endothelial nitric oxide synthase protein is not necessary for mechanical strain-induced nitric oxide production by cultured osteoblasts.
abstract: Regulation of nitric oxide (NO) production is considered essential in mechanical load-related osteogenesis. We examined whether osteoblast endothelial NO synthase (eNOS)-derived NO production was regulated by HSP90. We found that HSP90 is essential for strain-related NO release but appears to be independent of eNOS in cultured osteoblasts.
  
  
  BACKGROUND
  NO is a key regulator of bone mass, and its production by bone cells is regarded as essential in mechanical strain-related osteogenesis. We sought to identify whether bone cell NO production relied upon eNOS, considered to be the predominant NOS isoform in bone, and whether this was regulated by an HSP90-dependent mechanism.
  
  
  METHODS
  Using primary rat long bone-derived osteoblasts, the ROS 17/2.8 cell line and primary mouse osteoblasts, derived from wild-type and eNOS-deficient (eNOS(-/-)) mice, we examined by immunoblotting the expression of eNOS using a range of well-characterised antibodies and extraction methods, measured NOS activity by monitoring the conversion of radiolabelled L-arginine to citrulline and examined the production of NO by bone cells subjected to mechanical strain application under various conditions.
  
  
  RESULTS
  Our studies have revealed that eNOS protein and activity were both undetectable in osteoblast-like cells, that mechanical strain-induced NO production was retained in bone cells from eNOS-deficient mice, but that this strain-related induction of NO production was, however, dependent upon HSP90.
  
  
  CONCLUSIONS
  Together, our studies indicate that HSP90 activity is essential for strain-related NO release by cultured osteoblasts and that this is highly likely to be achieved by an eNOS-independent mechanism.
authors.affiliation: Department of Veterinary Basic Science, Royal Veterinary College, London, NW1 0TU, UK.
authors.forename: V
authors.initials: V
authors.lastname: Das-Gupta
authors.forename: R A
authors.initials: RA
authors.lastname: Williamson
authors.forename: A A
authors.initials: AA
authors.lastname: Pitsillides
mesh_terms: D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D002478:Cells, Cultured; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000502:physiology; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D040542:Mechanotransduction, Cellular / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000172:deficiency / Q000378:metabolism* / Q000502:physiology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013314:Stress, Mechanical
chemical_list: D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; C001277:geldanamycin