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Jennifer Uno
Ph.D. Candidate
Physiological Sciences
Digestive Disease Weak
Los Angeles, California
May 20-25, 2006
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“The Role of TNF? in Down-Regulation of Osteoblast Phex Gene Expression in Experimental Murine Colitis”
ABSTRACT
Background and Aims: Decreased bone mineral density is a common out come of inflammatory bowel disease (IBD) Several studies published over the past decade indicate that as many as 31-59% of adult IBD patients are classified as osteopenic while 5-41% are actually diagnosed with osteoporosis. The pathogenesis involved in bone loss coupled with IBD is not completely understood with disease state, Ca2+ and vitamin D3 deficiency, glucocorticoid treatment, decrease gonadal function and overall poor nutrition all implicated as possible causes. Phex gene is expressed predominantly in osteoblasts and its disruption results in abnormal phosphate handling as well as defective bone mineralization. The aim of this study was to evaluate whether TNF? regulates Phex gene expression thus contributing to the abnormal bone metabolism observed in IBD. Materials and Methods: Phex gene expression was evaluated in calvaria of 6-7 week old mice administered intrarectally with TNBS ( 2 mg in 100 ?L of 50% ethanol) or systemically (IP) with recombinant TNF? (15 ?L/100g BW, or PBS as control). In addition, Phex mRNA expression was evaluated in vitro in TNF?-treated UMR-106 osteosarcoma osteoblast-like cells. Phex gene promoter activity was assayed in transiently transfected UMR-106 cells treated with TNF?. Results: Real-time PCR results indicated a 40% decrease in Phex mRNA expression in TNBS- treated animals and a 50% decrease in mice directly injected with TNF?. Local attenuation of colitis and TNF? expression by dietary curcumin counteracted the detrimental effects of TNBS on Phex gene expression. UMR-106 cells showed decreasing Phex mRNA expression with increasing concentrations of TNF?, with a maximum reduction seen at 25ng/ml of TNF?. Transfection experiments with -1064/+104 nts of the Phex promoter construct showed a similar dose response curve. In order to determine the promoter region responsible for TNF?-regulation, cells were transfected with Phex promoter constructs -1064/+104, -542/+104, and -133/+104. Activity of all three promoter constructs were reduced approximately 60% with TNF? treatment. Western blot analysis from protein isolated from TNF?-treated UMR-106 cells revealed a decrease in Phex protein expression; mineralization of cultured UMR-106 cells was also drastically decreased with TNF? treatment. Conclusions: These studies are the first to demonstrate regulation of Phex gene expression in a mouse model of chemically induced colitis. TNF? appears to decrease Phex mRNA and protein expression via a transcriptional mechanism. TNF?-mediated decreases in Phex protein is at least in part, responsible for inhibition of osteoblast mediated-minerlaization. Collectively, this data suggests that Phex may contribute to the abnormal bone metabolism associated with IBD.
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