Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Oxidative stress contributes to osteoporosis by directly suppressing the development and function of osteoblasts, the cells that form new bone. Iron overload results in fewer mature osteoblasts and reduced matrix formation and mineralization in vitro, in part through inducing oxidative stress. Studies are now being conducted to examine the extent to which dietary antioxidant compounds can preventor minimize the impact of oxidative stress, such as what is induced by iron overload, on osteoblast maturation and function in cell culture. Quercetin is an antioxidant flavonoid found abundantly in plant-based foods, and appears to protect cells from oxidative stress by up-regulating stress signaling pathways. Therefore, quercetin is a viable target molecule to investigate the extent that dietary flavonoids prevent or reverse oxidative stress-related contributions in the development of osteoporosis. The primary objective of this research is to understand the extent that dietary quercetin impacts the oxidative stress response of osteoblasts and the development of the osteoblast phenotype in a high oxidative stress environment. The overall hypothesis is that quercetin will preserve development of the osteoblast phenotype when the cells are in a high oxidative stress environment by quercetin-mediated alterations in the osteoblast stress response pathway. Experiments were performed in primary osteoblast-like cells isolated from the calvaria (frontal and parietal bones) of fetal rats. Oxidative stress was induced in cell cultures by addition of hydrogen peroxide, which is a commonly used inducer of oxidative stress. Three quercetin metabolites that are commonly found in blood plasma after consumption of quercetin or quercetin-containing foods were used: quercetin aglycone, isorhamnetin, and quercetin-3-O-glucuronide. Analysis of multiple metabolites is essential since consuming quercetin from dietary sources results in enzymatic modification of the compound during digestion, and therefore multiple metabolites are found in the blood plasma. Cells were treated using each plasma metabolite separately(0-20 uM)or as a mixed dose (50:25:25 quercetin-3-O-glucuronide to isorhamnetin to quercetin aglycone). This mixed treatment group will evaluate potential synergistic effects between the metabolites. The impact of quercetin metabolites on the osteoblast phenotype was assessed by measuring osteoblast-specific gene markers, Runx2, alkaline phosphatase, bone sialoprotein, and osteocalcin using quantitative real-time PCR. Bone nodules were stained for alkaline phosphatase and mineralization using the von Kossa technique. Bone nodules were counted using an inverted microscope under bright-field illumination. Analysis of the impact of quercetin metabolites on the oxidative stress response is underway, using real-time PCR and Western blotting. PARTICIPANTS: Deborah Kipp, PI, developed overall research focus, study design, methods of analysis, interpretation of results. Mr. Jon Messer, PhD student in Nutrition, assisted in planning, conducting, and analyzing results of these studies. Ms. Stephanie La, undergraduate researcher, assisted in preparing solutions and collecting samples TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Experiments were conducted to establish the most effective way to induce oxidative stress in fetal rat calvaria. A dose response experiment was performed with 0-300 uM hydrogen peroxide to determine which dose effectively suppressed osteoblast phenotype without causing marked cell death. Results indicated that treating cells with 300 uM hydrogen peroxide throughout differentiation markedly suppressed alkaline phosphatase staining and mineralization compared to control (0 uM). Next, experiments were conducted to determine the time frame that differentiating cells were most sensitive to hydrogen peroxide-induced phenotype suppression. Cells treated with 300 uM hydrogen peroxide during the first 4 days of the differentiation period (days 5-9) showed less alkaline phosphatase positive staining compared to control. In contrast, cells treated with hydrogen peroxide later in culture were similar to control. Taken together, these results suggest that cells are most sensitive to 300 uM hydrogen peroxide during the first 4 days of differentiation (days 5-9). Next, experiments were conducted to determine the extent that the quercetin metabolites altered differentiation in fetal rat calvarial cultures. Cells treated with 0-10 uM quercetin metabolites throughout differentiation (days 5-21) exhibited similar alkaline phosphatase staining and mineralization compared to control on D21, suggesting that long-term treatment of quercetin metabolites does not adversely effect fetal rat calvarial cultures. Cells were also acutely treated for 48h during early differentiation (day 6-8) with 0-10 uM quercetin aglycone. This treatment did not significantly alter the expression levels of any osteoblast phenotypic genes. Furthermore, short-term treatment (48h) with up to 20 uM quercetin aglycone early in culture (days 6-8) did not alter alkaline phosphatase staining after 48h treatment. Thus, these metabolites did not negatively effect bone phenotypic development or function. Next, an experiment was designed to examine the extent to which quercetin metabolites alter oxidative stress pathway in osteoblasts. Cells were treated during early differentiation with quercetin metabolites either separately at 0-20 uM or with the 50:25:25 ratio of quercetin-3-O-glucuronide to isorhamnetin to quercetin aglycone. Analyses of genes and proteins that are known targets of quercetin and are involved in the oxidative stress pathway are underway. Additional studies will then be conducted to examine the protective effect of quercetin pre-treatment on osteoblast response to oxidative stress. Results of these studies will provide a greater understanding of how dietary antioxidants interact with and potentially protect bone cells under oxidative stress. This is the first study to be conducted in a primary osteoblast cell model using physiologically relevant doses of quercetin metabolites found in blood plasma. By including multiple metabolites in a range of physiological doses, the effects of dietary quercetin can be more completely assessed.
Publications
- No publications reported this period
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Iron overload and iron imbalance overall have been linked to low bone mass in humans. The impact of low to excessive intracellular iron status on bone cell function is poorly understood and is the focus of the current studies. These studies are being conducted using osteblast-like cells isolated from fetal rat calvaria. This is a well-established primary cell culture model of osteoblast phenotypic development and function. Cells are treated with either excessive iron levels (0-10 uM iron sulfate) throughout differentiation, or to a low intracellular iron environment that is achieved by treatment with desferoxamine (DFOM). Low iron status is achievable by treatment with DFOM which is a membrane-permeable iron chelator that prevents cellular iron update and chelates iron from the intracellular iron pool. Using this experimental model, the effect of DFOM on iron-regulated genes and proteins and the extent to which low iron availability affects osteobalst differentiation and function could be evaluated.Intracellular iron status was assessed by measuring iron content in cell layers and changes in transferrin receptor and ferritin gene and protein expression. Osteoblast differentiation and function were evaluated by measuring osteoblast phenotypic gene markers and capacity of cultures to form mineralized bone nodules. PARTICIPANTS: Dr. Deborah E.Kipp, Principle Investigator; developed overall research focus, study design, methods of analysis, interpretation of results. Mr. Jon G. Messer, Research Technician (switched to PhD student); assisted in study execution, analysis of samples, data analysis, and manuscript write-up. Ms. Amy Kilbarger, MS student; assisted in study execution, analysis of samples, and data analysis. Dr. Keith Erikson, Collaborator; expertise in cellular iron regulation, participated in discussions of study design, treatments to alter iron levels, and iron analysis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results of experiments conducted thus far suggest that with iron overload, iron accumulates in bone forming cells (osteoblasts) in culture. These studies were conducted using primary cultures of osteoblast-like cells isolated from fetal rat calvaria. Accumulation of high levels of intracellular iron, due to iron overload, is accompanied by alterations in gene expression and protein levels of iron-regulating proteins, transferrin receptor and ferritin. The associated shift in iron status is accompanied by decreased bone cell function (suppressed osteoblastic development and function) and cell death. Additional studies have been conducted to further explore the extent to which iron imbalance influences osteoblast phenotypic development and function. Nodule formation was similar to controls with the addition of up to 4 uM DFOM throughout differentiation. Expression of osteoblastic genes that are indicative of development of the osteoblast phenotype (bone sialoprotein and osteocalcin) were lower than normal at DFOM levels of 4 uM or higher, indicating that iron is needed for normal osteoblast development. Bone nodule formation, a functional indicator of osteoblast activity, was markedly suppressed with both 8 uM DFOM (67% lower) and 32 uM DFOM (77% lower), suggesting that osteoblast function is also suppressed with iron deficiency. DFOM resulted in a greater reduction of nodule formation during early differentiation (d8-15; 45% lower) than later differentiation (d15-21; 17% lower), suggesting that there is a critical timing in the requirement for iron. In addition, 8 uM DFOM was the lowest dose to maximally suppress osteoblast phenotypic development (based on the combined gene analysis and functional analysis of bone nodule formation).
Publications
- Messer, J.G., Kilbarger, A.K., Erikson, K.M., Kipp, D.E. (2009) Iron overload alters iron-regulatory genes and proteins, down-regulates osteoblastic phenotype, and is associated with apoptosis in fetal rat calvaria cultures. Bone 45(5):972-9.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Iron overload has been implicated in decreased bone mineral density. However, the effect of iron status on cells of the osteoblast lineage remains poorly understood. Therefore, the purpose of this study was to examine differentiation and function of iron loaded osteoblast-like cells isolated from fetal rat calvaria. Since iron overload is associated with cytotoxicity, apoptosis was also monitored. Fetal rat calvaria cultures are a well-established model of osteoblast differentiation, characterized by temporal upregulation of osteoblast phenotype genes and emergence of multilayered, mineralized nodules resembling woven bone. Cells were exposed to concentrations of FeS (0 - 10 uM) throughout differentiation (day 6 - day 20). Samples were taken during acute (up to 48 hours) and chronic (up to 2 weeks) exposure to FeS. Iron concentration in the cell layer was measured with graphite furnace atomic absorption spectrometry. Intracellular iron loading was confirmed by assessing alteration in gene and protein expression of transferrin receptor (TrfR) and ferritin light (FerL) and heavy (FerH) subunits using RT-PCR and Western blotting. Osteoblast phenotype gene markers were analyzed with RT-PCR using primers for osteocalcin, bone sialoprotein, alkaline phosphatase, and collagen 1a. The functional capacity of the cultures was reflected in the number of mineralized nodules counted at the end of the differentiation period. Apoptotic cells were observed with microscopy after Annexin V-fluorescein staining or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to discern translocation of phosphatidyl serine to the outer leaflet of cell membranes and oligonucleosomal DNA fragmentation, respectively. PARTICIPANTS: Deborah E. Kipp, Principal Investigator; developed overall research focus, study design, methods of analysis, interpretation of results Jon G. Messer, Research Technician; assisted in study execution, analysis of samples, and data analysis TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The 5 uM FeS treatment in this study resulted in a 2-fold increase in iron in the media, similar to serum iron levels observed in humans with hemochromatosis and iron-loaded animals with altered bone metabolic parameters. The 5 uM treatment appeared to saturate iron content in cell layers 20-fold higher than control by mid-differentiation (day 15) (p less than 0.05). Lower iron doses (1 uM) had no significant effect on iron content or alteration of TrfR or ferritin genes or proteins after chronic exposure. Alteration of TrfR gene and protein expression and ferritin protein expression confirm that iron accumulated intracellularly. Acute exposure to FeS resulted in rapid down-regulation of TrfR genes with marked protein down-regulation observed after 24 hours. Chronic exposure to 5 uM FeS sustained suppression of TrfR protein and up-regulated FerL and FerH proteins by day 20. Intracellular iron loading was associated with suppression of osteoblast differentiation and function, and apoptosis. Cultures treated chronically with 5 or 10 uM FeS exhibited suppressed phenotype gene markers and lower numbers of mineralized nodules. Unmineralized nodules were present in all treatments, and the total number of nodules (mineralized and unmineralized) did not significantly decrease with iron treatment. Apoptotic events were observed within 24 hours after 5 uM and 10 uM FeS treatments and were similar to those observed in the hydrogen peroxide treated positive control. Apoptotic cells in iron treated cell layers were generally located within and surrounding multilayered areas. This is the first report describing iron accumulation in osteoblasts cultured in iron overload conditions in vitro. The corresponding alteration in TrfR and ferritin confirm that osteoblastic cells accumulate iron when extracellular iron is present in excess. The associated shift in iron status appears to be associated with cytotoxicity characteristic of apoptosis and suppressed osteoblast differentiation and function, consistent with decreased bone mineral density in vivo.
Publications
- No publications reported this period
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