Discovery of Novel Vitamin D Receptor Interacting Proteins (VIPs) Involved in Apoptosis and Bone Mineral Homeostasis
Lemau, Jana L.; Marshall, Pamela A.; and Jurutka, Peter
Department of Integrated Natural Sciences, Arizona State University, West Campus
The nuclear vitamin D receptor (VDR) is known to regulate transcription of vitamin D responsive genes involved in bone mineral homeostasis. Runx2, a critical protein required for bone development, and TFIIB, a transcription factor recruited by VDR, have been implicated in bone signaling as VDR interacting proteins (VIPs). We are evaluating the physical interaction between VDR and potential VIPs, including Runx2 and TFIIB, using GST-based interaction assays. These GST pulldown assays demonstrated direct vitamin D-independent binding of VDR with TFIIB and Runx2. We are also utilizing the yeast two hybrid system to discover novel potential VIPs. L40 yeast cells were transformed with a bait (LEXA-VDR) fusion construct and a prey library. Eleven potential VIPs were identified and analyzed by sequence analysis. 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 match. Further GST-based interaction assays were performed with VDR, RXR and radiolabeled APITD1. These assays revealed a direct association of VDR and APITD1, with RXR acting as a potential inhibitor of this interaction. TFIIB, Runx2, RANKL, and APITD1 genes were cloned into M2H vectors for further evaluation of VDR interaction in osteoblasts.