This is a competing renewal application designed to continue studies on phosphate transport and homeostatic mechanisms during health and disease states. This renewal will focus on bone disease associated with inflammatory bowel disease (IBD) as it relates to phosphate homeostasis. To accomplish this goal, we are planning a series of in vitro and in vivo experiments to gain a deeper understanding of the mechanisms governing phosphate homeostasis, the gut-renal-skeletal axis, and immune-skeletal interactions in IBD at systemic, cellular and molecular levels. The field on phosphate homeostasis has recently seen major advances which challenge many of the established concepts. These advances include the discovery of phosphatonins and the Klotho gene. Therefore the central hypothesis to be tested in this proposal is that in acute and chronic colitis, TNFa is a central player in the disruption of the gut-skeletal- renal axis responsible for inorganic phosphate homeostasis and proper bone mineralization. To address this hypothesis, we are planning to investigate three specific aims: 1. To identify molecular mechanisms of TNFa-mediated down-regulation of Phex gene expression in osteoblasts. The hypothesis of this aim is that Phex, a gene indispensable for osteoblast mineralization and indirectly participating in inorganic phosphate reabsorption, is significantly down-regulated in acute and chronic colitis. TNFa mediates decreased Phex gene transcription through a novel molecular mechanism involving a poly-adenine region of the proximal Phex gene promoter. 2. To describe molecular mechanisms of downregulation of sodium-phosphate co-transporter (NaPi-IIa) expression at the transcriptional and post-translational levels by inflammatory mediators in acute and chronic colitis. The hypothesis which will be addressed in this specific aim is that in both acute (chemically induced) and chronic (in IL-10-/- mice) colitis, TNFa affects expression and cellular distribution of NaPi-IIa via distinct mechanisms involving protein trafficking and/or degradation and gene transcription. 3. To determine the effects of colitis and the role of TNFa in the regulation of Klotho gene expression. Here, we will investigate in vivo the effects of intestinal inflammation on expression of Klotho mRNA and protein, and characterize its endogenous expression and transcriptional regulation in vitro in immortalized mouse renal epithelial cells of distal convoluted tubules by TNFa. These studies are novel and will certainly lead to a better understanding of the effects of intestinal inflammation on the process of phosphate homeostasis as it relates to bone mineralization in disease states.
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