In vitro analysis of osteoblasts deficient in endothelial NO synthase (eNOS) or inducible NOS (iNOS) enzymes, indicated that NO produced via eNOS is required for normal osteoblast growth and differentiation. The transcriptional regulation of two genes, the extracellular matrix protein Tenascin-Cytotactin (Tn-C) and eNOS, which contain strain-related regulatory sites and are known to respond to mechanical stimuli, was investigated in osteoblasts and osteocytes (ob-mix). Stretch was applied via 2 different systems and the transcription level of the gene promoters assayed by dual luciferase reporter assay. It was demonstrated that transcription of Tn-C and eNOS is upregulated in response to physiological levels of tensile and/or shear stress. This signal is mediated via the putative StRE and SSRE in Tn-C promoter and at the proximal end of the eNOS promoter is indicated. A system was built to study calcium (Ca<sup>2+</sup>) fluxes in response to shear stress in primary ob-mix and an immortalised simian virus human foetal osteoblast cell-line (SV-HFO). Studies have shown that pulsating fluid flow (PFF) stimulates a rapid [Ca<sup>2+</sup>]<sub>i</sub> increase in primary ob-mix. Rapid transient increases in [Ca<sup>2+</sup>]<sub>i</sub> were also recorded in a synchronised culture of SV-HFO when subjected to PFF. Both these observations are consistent with the hypothesis that Ca<sup>2+</sup> increases contribute to the osteoblastic NO response to shear stress. Taken together, these results confirm the important role NO plays in bone physiology and elucidate aspects of transcriptional regulation and activation of the eNOS enzyme.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:369737 |
| Date | January 2001 |
| Creators | Gallagher, Marie |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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