Meeting Abstract
P2.92 Jan. 5 Characterization and Functional Evaluation of Novel Vitamin D Receptor Interacting Proteins (VIPs) Required for Bone Development LEMAU, Jana L.*; MARSHALL, Pam A.; JURUTKA, Peter W.; Arizona State University West Campus; Arizona State University West Campus; Arizona State University West Campus Lemaumau14@yahoo.com
The vitamin D receptor (VDR) is known to regulate transcription of genes such as those involved in calcium homeostasis and/or bone formation. &beta-Catenin, a mediator of canonical Wnt signaling, has been implicated in bone development and may act as a VDR interacting protein (VIP). In this study, we evaluated the physical and functional association between VDR and &beta-catenin. We first characterized this interaction in vitro and in human osteosarcoma cells using GST-based interaction assays and the mammalian two hybrid (M2H) system. The GST pulldown assays demonstrated a direct association between GST-VDR and 35S-radiolabeled &beta-catenin or GST-&beta-catenin and labeled VDR. M2H assays confirmed the &beta-catenin-VDR association in human osteoblasts. &beta-catenin truncation mutants were constructed and tested for interaction with VDR to map the region of &beta-catenin required for VDR association. Finally, we employed the yeast two hybrid (Y2H) approach to discover novel potential VIPs. Eleven potential VIPs isolated via Y2H were evaluated by sequence analysis, and clones 5, 6, and 8 were found to be of potential significance. A protein BLAST search of the mammalian database with Clone 6 resulted in a significant match to RANKL, a cell-surface receptor that controls osteoclastogenesis and calcium metabolism. Analysis of clones 5 and 8 revealed apoptosis-inducing TAF9-like domain 1 (APITD1) as a distinct match. These proteins are currently being evaluated in GST pulldown and M2H assays to confirm their specific interaction with VDR. Identification of -catenin, RANKL and APIT as significant VIPs may provide novel mechanistic insights into the role of VDR in bone development/mineral homeostasis and vitamin D-mediated cellular apoptosis.
