Endothelial nitric oxide synthase is not essential for nitric oxide production by osteoblasts subjected to fluid shear stress in vitro

Astrid D. Bakker, Carmen Huesa, Alun Hughes, Richard M. Aspden, Rob J. van't Hof, Jenneke Klein-Nulend, Miep Helfrich

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Endothelial nitric oxide synthase (eNOS) has long been held responsible for NO production by mechanically stimulated osteoblasts, but this has recently been disputed. We investigated whether one of the three known NOS isoforms is essential for NO production by mechanically stimulated osteoblasts in vitro and revisited the bone phenotype of the eNOS(-/-) mouse. Osteoblasts, obtained as outgrowths from mouse calvaria or long bones of wild-type (WT), eNOS(-/-), inducible NOS(-/-) (iNOS(-/-)), or neuronal NOS(-/-) (nNOS(-/-)) mice, were subjected to mechanical stimulation by means of pulsating fluid flow (PFF); and NO production was determined. Tibiae and femora from 8-week-old mice were subjected to µCT and three-point bending tests. Deletion of single NOS isoforms did not lead to significant upregulation of alternate isoforms in cultured osteoblasts from WT, eNOS(-/-), iNOS(-/-), or nNOS(-/-) mice. Expression of eNOS mRNA in osteoblasts was below our detection limit, and no differences in growth between WT and eNOS(-/-) osteoblasts were found. PFF increased NO production by approximately fourfold in WT and eNOS(-/-) osteoblasts and significantly stimulated NO production in iNOS(-/-) and nNOS(-/-) osteoblasts. Tibiae and femora from WT and eNOS(-/-) mice showed no difference in bone volume and architecture or in mechanical parameters. Our data suggest that mechanical stimuli can enhance NO production by cultured osteoblasts singly deficient for each known NOS isoform and that lack of eNOS does not significantly affect bone mass and strength at 8 weeks of age. Our data challenge the notion that eNOS is a key effector of mechanically induced bone maintenance.
Original languageEnglish
Pages (from-to)228-239
Number of pages12
JournalCalcified Tissue International
Volume92
Issue number3
Early online date1 Dec 2012
DOIs
Publication statusPublished - Mar 2013

Fingerprint

Nitric Oxide Synthase Type III
Osteoblasts
Nitric Oxide
Protein Isoforms
Bone and Bones
Pulsatile Flow
Tibia
Femur
In Vitro Techniques
Skull
Limit of Detection
Up-Regulation
Maintenance
Phenotype
Messenger RNA

Keywords

  • inducible nitric oxide synthase
  • endothelial nitric oxide synthase
  • neuronal nitric oxide synthase
  • mechanical stimulation
  • osteoblast

Cite this

Endothelial nitric oxide synthase is not essential for nitric oxide production by osteoblasts subjected to fluid shear stress in vitro. / Bakker, Astrid D.; Huesa, Carmen; Hughes, Alun; Aspden, Richard M.; van't Hof, Rob J.; Klein-Nulend, Jenneke; Helfrich, Miep.

In: Calcified Tissue International, Vol. 92, No. 3, 03.2013, p. 228-239.

Research output: Contribution to journalArticle

Bakker, Astrid D. ; Huesa, Carmen ; Hughes, Alun ; Aspden, Richard M. ; van't Hof, Rob J. ; Klein-Nulend, Jenneke ; Helfrich, Miep. / Endothelial nitric oxide synthase is not essential for nitric oxide production by osteoblasts subjected to fluid shear stress in vitro. In: Calcified Tissue International. 2013 ; Vol. 92, No. 3. pp. 228-239.
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AU - van't Hof, Rob J.

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N2 - Endothelial nitric oxide synthase (eNOS) has long been held responsible for NO production by mechanically stimulated osteoblasts, but this has recently been disputed. We investigated whether one of the three known NOS isoforms is essential for NO production by mechanically stimulated osteoblasts in vitro and revisited the bone phenotype of the eNOS(-/-) mouse. Osteoblasts, obtained as outgrowths from mouse calvaria or long bones of wild-type (WT), eNOS(-/-), inducible NOS(-/-) (iNOS(-/-)), or neuronal NOS(-/-) (nNOS(-/-)) mice, were subjected to mechanical stimulation by means of pulsating fluid flow (PFF); and NO production was determined. Tibiae and femora from 8-week-old mice were subjected to µCT and three-point bending tests. Deletion of single NOS isoforms did not lead to significant upregulation of alternate isoforms in cultured osteoblasts from WT, eNOS(-/-), iNOS(-/-), or nNOS(-/-) mice. Expression of eNOS mRNA in osteoblasts was below our detection limit, and no differences in growth between WT and eNOS(-/-) osteoblasts were found. PFF increased NO production by approximately fourfold in WT and eNOS(-/-) osteoblasts and significantly stimulated NO production in iNOS(-/-) and nNOS(-/-) osteoblasts. Tibiae and femora from WT and eNOS(-/-) mice showed no difference in bone volume and architecture or in mechanical parameters. Our data suggest that mechanical stimuli can enhance NO production by cultured osteoblasts singly deficient for each known NOS isoform and that lack of eNOS does not significantly affect bone mass and strength at 8 weeks of age. Our data challenge the notion that eNOS is a key effector of mechanically induced bone maintenance.

AB - Endothelial nitric oxide synthase (eNOS) has long been held responsible for NO production by mechanically stimulated osteoblasts, but this has recently been disputed. We investigated whether one of the three known NOS isoforms is essential for NO production by mechanically stimulated osteoblasts in vitro and revisited the bone phenotype of the eNOS(-/-) mouse. Osteoblasts, obtained as outgrowths from mouse calvaria or long bones of wild-type (WT), eNOS(-/-), inducible NOS(-/-) (iNOS(-/-)), or neuronal NOS(-/-) (nNOS(-/-)) mice, were subjected to mechanical stimulation by means of pulsating fluid flow (PFF); and NO production was determined. Tibiae and femora from 8-week-old mice were subjected to µCT and three-point bending tests. Deletion of single NOS isoforms did not lead to significant upregulation of alternate isoforms in cultured osteoblasts from WT, eNOS(-/-), iNOS(-/-), or nNOS(-/-) mice. Expression of eNOS mRNA in osteoblasts was below our detection limit, and no differences in growth between WT and eNOS(-/-) osteoblasts were found. PFF increased NO production by approximately fourfold in WT and eNOS(-/-) osteoblasts and significantly stimulated NO production in iNOS(-/-) and nNOS(-/-) osteoblasts. Tibiae and femora from WT and eNOS(-/-) mice showed no difference in bone volume and architecture or in mechanical parameters. Our data suggest that mechanical stimuli can enhance NO production by cultured osteoblasts singly deficient for each known NOS isoform and that lack of eNOS does not significantly affect bone mass and strength at 8 weeks of age. Our data challenge the notion that eNOS is a key effector of mechanically induced bone maintenance.

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KW - endothelial nitric oxide synthase

KW - neuronal nitric oxide synthase

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KW - osteoblast

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