Bone Mineralization in a Murine Model of Inflammatory Bowel Disease

Abstract

Reduced bone mass is a common complication of human inflammatory bowel disease, however, the mechanisms that contribute to osteopenia are not completely understood. Cytokines are up regulated in IBD patients and have been shown to have detrimental effects on osteoblasts. PHEX is expressed predominantly in osteoblasts; disruption of the PHEX gene results in defective bone mineralization and renal phosphate wasting. We hypothesize that PHEX gene expression as well as overall Pi homeostasis are altered in individuals with IBD and therefore, may contribute to alterations in bone mineral density observed in individuals with IBD. In vivo studies: 6-7 week old Balb/C mice were intrarectally instilled with TNBS or 50% ethanol. Animals were treated with or without neutralizing anti-TNFα antibody, dietary curcumin, or systemically with recombinant TNFα. RNA was prepared from bone and gene expression was analyzed by PCR. Kidney and small bowel were harvested from control and TNBS treated animals and embedded in paraffin for immunohistochemical analysis. In vitro studies: Cells were treated with IFNγ, IL1-β, IL-6, or TNFα and RNA was collected for real-time PCR analysis. UMR-106 cells were transfected with Phex promoter constructs. PHEX is down-regulated in mice with chemically induced colitis and in the mice injected with TNFα, this decrease was attenuated by curcumin and TNFα antibody. TNFα decreased endogenous levels of PHEX mRNA, protein, and inhibited spontateous mineralization in UMR-106 cells. This regulation occured at a transcriptional level and it appears that the proximal poly-A region of the PHEX promoter played a significant role in downregulation of Phex expression. Phosphatonin expression was not altered in TNFα-treated UMR-106 osteoblasts. Cytokines were unable to alter phosphatonin mRNA expression in UMR-106 cells and no changes in phosphatonin expression were observed in vivo. Intestinal NaPi-IIb mRNA expression decreased in TNBS-treated animals although immunohistochemical analysis did not reveal any changes in cellular localization of NaPi-IIb protein. Renal NaPi-IIa mRNA did not change in TNBS-treated animals however, immunohistochemical analysis revealed internalization of NaPi-IIa from the apical membrane. In conclusion, decreased phosphate absorption in the kidney along with altered Phex gene expression may contribute to decreased bone mineral density observed in IBD patients.