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Title: Role of Oxidative Stress In Bone Loss And Gene Expression In Osteoblasts
Author: Varanasi, Satya Srinivas
ISNI:       0000 0001 3542 6763
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2004
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Mitochondrial DNA (mtDNA) deletions disrupt electron transport chain and increase the generation of reactive oxygen species (ROS). Mitochondria are both a major source of oxidants and a target for their damaging effects; mitochondrial oxidative stress appears to be a cause of cell aging and damage. Mitochondrial DNA deletions (dmtDNA) accumulate with age and are responsible for the failure of function and ensuing of disease in a variety of tissues. Bone is also a tissue that is known to be associated with agerelated changes. Despite the obvious association of changes in bone with ageing, the role of dmtDNA and resultant oxidative stress in bone has never been studied. In light of above, we screened men with symptomatic vertebral fractures and agematched control men for mtDNA deletions in blood leucocytes using polymerase chain reaction (PCR) and Southern blotting. PCR based analysis revealed the presence of the common age-related 4.9 Kb dmtDNA in 9 out of 15 (60%) men with symptomatic vertebral fractures and 5 out of the 17 (29) male control subjects. However, Southern blotting revealed evidence of 3.7 Kb mtDNA deletions in only 2 patients and none in c'ontroI male subjects. The patient with highest percentage (~50%) of 3.7 Kb dmtDNA was a 27 yr old man with very severe osteoporosis' Following the above observation, the dmtDNA screening using Southern blotting was carried out on cortical bone biopsies obtained from subjects undergoing hip and knee arthroplasty. Three overlapping probes (5Kb, 8.4 Kb and 12 Kb) spanning the entire mitochondrial genome were used to screen 30 specimen. No mtDNA deletions were observed in the investigations using the 5Kb and 8.4Kb probes. However, screening with the 12Kb probe revealed variable levels of deletions in 20 out of 30 specimens. Six patients displayed high levels of deletions, while moderate amounts of deletions were observed in another 14 subjects. The most prominent deletions were 1.1 Kb foHowed by 5 Kb in 4 biopsy specimen; other evident minor deletions were 3.2 Kb, 9Kb, 6Kb and 10Kb fragments. The existence of extensive accumulation of age-related dmtDNA and its implication for local oxidative stress bone microenvironment led to us examine the effect of hydrogen peroxide on gene regulation in osteoblast-like cell line (ROS 17/2.8 cells). We employed differential display reverse transcriptase-PCR to screen modulated mRNA transcripts from ROS 17/2.8 cells transiently exposed to hydrogen peroxide. The mRNA transcripts that were dramatically induced or suppressed following transient exposure to hydrogen peroxide were isolated and sequenced. A vast majority of the clones that were sequenced were found to be known sequences whilst a few were novel; a known and a novel sequence were selected for in-depth investigation. 3' UTR region of one of the novel gene selected for in-depth investigation had ful 81.9% sequence identity with rat T-cell receptor (TCR) and 86.6 % homology with a murine gene of unknown function located on chromosome 10. Northern blotting revealed three mRNA transcripts; the largest of which (~1.4 Kb) was observed most prominently in bone, testes, heart and kidney; the intermediate (~1.2 Kb) transcript was fuund only in bone; the smallest (~1.1 Kb) was present lung, bone and testes. The osteoblast-like osteosarcoma cells (ROS 17/2.8) were found to express only the larger mRNA transcript. Domain analysis revealed 10 out 14 similar domains between our molecule and rodent, murine and human TCR sequences; suggesting the novel molecule is a homologue of TCR (TCRh). \Vestern blotting with anti-rat TCR monoclonal antibody (R73), revealed a ~45 kDa TCR homologue (TCRh) in ROS 17/2.8 cell lysate. The exposure of rat osteoblast-like cells to parathyroid hormone (PTH) and 1, 25a dihydroxycholecalciferol (vitamin D3) markedly suppressed the TCRh expression; with parathyroid hormone being more inhibitory than vitamin D3. lL-6 release was modulated by the monoclonal antibody (R73); when used together with a cross-linker was found to be substantially more potent than PTH in affecting release of IL-6. Interestingly osteoprotegerin (OPO) mRNA expression in the R73 stimulated cells, was found to be up-regulated following 18 to 24 hour incubation with the R73 antibody and was also found to be sustained at 48 hours revealed by Northern blotting. An immunophilin FK506 binding protein 12 (FKBPI2) mRt''J'A expression was upregulated by hydrogen peroxide and this was also selected for in-depth study. FKBP12 is expressed in a variety of tissues but its expression in osteoblast has never been demonstrated. We were able to demonstrate the expression of FKBP12 in both primary rat osteoblast and ROS 17/2.8 cells, and also detected a novel 10 kDa isoform (FKBPI 0) in these cells. In ROS 17/2.8 cells, vitamin D3 almost abolished expression of the 10 kDa isoform, PTH down-regulated the 12 kDa isoform while maintaining near basal level of 10 kDa protein expression. Hydrogen peroxide led to slight stimulation of both isoforms. The expression of FKBP12 and FKBPIO proteins in primary rat osteoblasts remained unaffected by the treatments with various agents. Following 24h exposure to anti-rat TCR antibody (R73), the expression of the smaller 10kDa isoform was suppressed and the heavier 12 KDa protein was significantly stimulated. The general importance of these observations and their wider implication III bone pathophysiology is discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available